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| Status |
Public on Aug 19, 2022 |
| Title |
Single Cell RNA-Sequence Analyses Reveal Uniquely Expressed Genes and Heterogeneous Immune Cell Involvement in the Rat Model of Intervertebral Disc Degeneration |
| Organism |
Rattus norvegicus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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
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| 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).
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| Contributor(s) |
Rohanifar M, Clayton SW, Easson G, Patil D, Lee F, Jing L, Barcellona M, Speer J, Stivers J, Tang S, Setton L |
| Citation(s) |
36451894 |
| Submission date |
Aug 16, 2022 |
| Last update date |
Jan 03, 2023 |
| Contact name |
Sade Williams Clayton |
| E-mail(s) |
sade@wustl.edu
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| Organization name |
Washington University in St. Louis
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| Department |
Biomedical Engineering
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| Street address |
1 Brookings Dr
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| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63130 |
| Country |
USA |
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| Platforms (1) |
| GPL25947 |
Illumina NovaSeq 6000 (Rattus norvegicus) |
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| Samples (16)
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| Relations |
| BioProject |
PRJNA870143 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE211407_RAW.tar |
431.8 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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