## 3 Results
### Cell Populations Identified in IVD Tissues from 2 Weeks Post-Surgery
Two representative samples are shown—(**a**) sample 3 (left) and (**b**) sample 4 (right) (Red dots represent genes expressed at higher levels in the LDP group while blue dots represent genes with higher expression levels in CON group) (**c**) Venn diagram represents the total number of commonly upregulated and downregulated genes for LDP and CON cells in across multiple samples (i.e., samples 3 and 4 here). (**d**) Heatmap showing the pattern for a subset of commonly upregulated genes with LDP compared to CON groups as a function of cell grouping (x-axis). Figure 5: The identification of immune cell subtypes in the myeloid and lymphoid clusters. (**a**) Diagram showing the differences in cell numbers mapped to the myeloid and lymphoid clusters between the control (CON) and degenerated (LDP) samples at two weeks after surgery. (**b**) Pan-macrophage marker expression and cell count differences between CON and LDP groups were used to confirm the identification of cells in the myeloid cluster. Macrophage polarization was assessed by identifying the presence of either (**c**) M1 or (**d**) M2 polarization cell surface markers, and the number of cells that expressed them. The presence of (**e**) B and (**f**) T lymphocytes was identified in the lymphoid cluster by measuring pan and subtype-specific marker expression levels and cell count changes. Numbers underneath each plot denote the number of cells expressing each marker. A Student’s \(t\)-test or a Mann–Whitney test was run to determine significance of marker expression levels. * = \(<\) 0.05, ** = \(<\)0.01, *** = \(<\)0.001, *** = \(<\)0.0001. For the lymphoid clusters, we likewise performed statistical analysis for the B lymphocytes (_Cd19_, _Cd79b_, _Blnk_, _Cd38_, _Cxcr4_) and T lymphocytes (_Cd3g_, _Cd4_, _Cd8b_) markers by assessing the number of cells that expressed them (Figure 5e,f). B cell receptor and co-receptor, _Cd79B_ and _Cd19_ in LDP samples, showed greater levels of gene expression compared to CON cells, and were expressed in the majority of the lymphoid cells (Figure 5e). Markers of B lymphocyte subtypes, _Blnk_, _Cd38_, and _Cxcr4_, all showed significantly greater gene expression in LDP samples concurrent with large increases in cell numbers between CON and LDP at 2 weeks after surgery (Figure 5e). As for T lymphocytes, the gene expression and numbers of cells expressing pan T cell markers, _Cd3g_, the T cell receptor, were both significantly increased (Figure 5f). A small number of cells expressed _Cd4_ or _Cd8b_ subtype markers with significant increases in gene expression (Figure 5f). Differential _Ngf_ expression in IVD cells in LDP populations. The number and marker expression of cells mapping to the IVD subclusters were evaluated between CON and LDP populations at 2 weeks after surgery. The number of cells expressing _Ngf_ and _Ngf_ (p75 NTR) and their relative expression were used to compare the IVD subclusters including the NP, IAF and OAF (Figure 5a,b). The Gene ontology functions ranked from top to bottom by enrichment score derived from [PERSON]'s exact test. The five most highly enriched processes identified by the Gene Set analyses were related to immune responses and associated regulatory processes (Figure 6c), consistent with the known infiltration of monocytes into the injured IVD post-surgical insult [41; 42; 43]. Genes associated with the top three regulated processes were upregulated in LDP samples and that are large drivers of the observed results are _Cd19_, _Cd37_, _Rag1_, _Blnk_, and _Myb_, as T- and B-cell associated marker genes. While few differences were observed in relative expression levels of _Ngf_ across the subclusters for the LDP group, the number of cells expressing _Ngf_ increased following injury (_p_ < 0.00001; Figure 6a). In CON cells from the NP cell sub-cluster, just 7.9% of cells expressed _Ngf_ which increased to 12.9% following injury (_p_ < 0.00001). _Ngf_ is believed to be important for promoting neurite infiltration into the otherwise avascular and aneural tissues of the NP [38].