## 3 Results
### Cell Populations Identified in IVD Tissues from 2 Weeks Post-Surgery
Differential gene expression between LDP and CON samples on a subject-specific basis. To directly assess differences in mRNA levels for cells from the CON (L2-3 and L3-4 levels) and LDP (L4-5 and L5-6 levels) groups within each rat sample, we performed specimen-specific differential gene expression (Figure 4a,b). Differential gene analyses demonstrated that between 700-900 genes were more highly expressed in LDP cells at 2 weeks after surgery, as compared to their counterparts amongst the CON cell population (FDR _p_-value < 0.001, Figure 4c). Analyses also showed between 250 and 1200 genes to be more highly expressed in CON cells as compared to the LDP cell population at 2 weeks after surgery (FDR _p_-value < 0.001, Figure 4c). While there was variability in differentially expressed genes across samples, we identified a subset of 25 commonly upregulated genes in LDP populations of cells from two samples with the highest total numbers of sequenced cells (i.e., samples #3 and #4); expression levels for these genes against their cell identification are shown in the heat map in Figure 4d (representative results shown for sample #4). Of these 25 genes with upregulated expression levels in LDP compared to CON cells, the majority (22/25) were associated with cells of the lymphoid or myeloid cell clusters (Figure 4d). Immune cell involvement in differences between LDP and CON gene expression patterns. We determined that immune cells were contributing to the upregulation genes due to LDP. Then, we classified the immune cell sub-types as either the myeloid or lymphoid clusters. There were significant increases in the number of cell mapping to the myeloid and lymphoid clusters for LDP samples as compared to CON samples at 2 weeks after surgery (_p_ < 0.00001; Chi-square test), indicating an increase of infiltrating immune cells post-injury. The lymphoid cluster showed the largest difference in cell number between CON and LDP conditions, where the LDP samples had a cell count larger than 4.6\(\times\) that of CON samples (_p_ < 0.00001; Figure 5a). For the myeloid cells, we performed statistical analysis on the expression level of the pan-macrophage markers (_Cd4_, _Cd14_, _Cd88_, and _Lyz2_), M1 polarization (_Tlr2_, _Tlr4_, _Cd80_, and _Cd86_), and M2 polarization (_Arg1_, _Arc1_ (_Cd206_), _Mrs1_ (_Cd204_)) cell surface markers (Figure 5b-d). The number of cells expressing pan-macrophage markers (_Cd4_, _Cd14_, _Cd68_, and _Lyz2_), as well as their respective levels of expression, indicated increased macrophages and heightened activity (Figure 5b). Furthermore, the number of cells expressing the canonical M1 cell surface markers _Tlr2_, _Tlr4_, and _Cd80_ were not  
Figure 3: (**a**) UMAP plot representation of five cell types within the LDP group at two weeks after surgery. (**b**) The expression level of marker genes within intervertebral disc cells, endothelial cells, myeloid, and lymphoid cells in LDP groups at two weeks after surgery. (**c**) Percentage of all analyzed cells mapped to each cell type for CON and LDP groups at two weeks after surgery. Inset provided to highlight apparent differences in CON and LDP groups for immune cell populations. different between groups, but the number of _Cd86+_ cells (_p_ < 0.00001) and their expression were higher in the LDP group (Figure 5c) [39]. Despite the similarity in the number of cells expressing M2 polarization markers between LDP and CON groups, including _Arg1_, _Mrc1_ (_Cd206_), and _Mrs1_ (_Cd204_), the expression levels were either indistinguishable (_Arg1_, _Cd206_) or lower (_Cd204_) compared with the CON group (Figure 5d) [39; 40]. Figure 4: Volcano plot depicting specimen-specific differenti gene expression between CON and LDaLP cells at two weeks after surgery.