Microarray analysis of laser capture microdissected-anulus cells from the human intervertebral disc.
METHODS
Five Thompson Grade I/II discs (Group 1), 7 Grade III discs (Group 2), and 3 Grade IV discs (Group IV) were studied here in a project approved by the authors' Human Subjects Institutional Review Board.


OBJECTIVE
Our objective was to use laser capture microdissection (LCM) to harvest cells from the human anulus and to derive gene expression profiles using microarray analysis.


BACKGROUND
Appropriate gene expression is essential in the intervertebral disc for maintenance of extracellular matrix (ECM), ECM remodeling, and maintenance of a viable disc cell population. During disc degeneration, cell numbers drop, making gene expression studies challenging.


METHODS
LCM was used to harvest cells from paraffin-embedded sections of human anulus tissue. Gene profiling used Affymetrix GeneChip Human X3P arrays. ANOVA and SAM permutation analysis were applied to dCHIP normalized, filtered, and log-transformed gene expression data ( approximately 33,500 probes), and data analyzed to identify genes that were significantly differentially expressed between the 3 groups.


RESULTS
We identified 47 genes that were significantly differentially expressed between the 3 groups (P < 0.001 and lowest q values). Compared with the healthiest discs (Grade I/II), 13 genes were up-regulated and 19 down-regulated in both the Grade III and the Grade IV discs. Genes with biologic significance regulated during degeneration involved cell senescence, low cell division rates, hypoxia-related genes, heat-shock protein 70 interacting protein, neuropilin 2, and interleukin-23p19 (interleukin-12 family).


CONCLUSIONS
Results expand our understanding of disc aging and degeneration and show that LCM is a valuable technique that can be used to collect mRNA amounts adequate for microarray analysis from the sparse cell population of the human anulus.
Gruber
Helen E
HE
Department of Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC 28232, USA. helen.gruber@carolinashealthcare.org
Mougeot
Jean-Luc
JL
Hoelscher
Gretchen
G
Ingram
Jane A
JA
Hanley
Edward N
EN
D000328:Adult; D002469:Cell Separation / Q000379:methods; D002574:Cervical Vertebrae; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007403:Intervertebral Disc / Q000737:chemistry / Q000473:pathology*; D007834:Lasers; D008159:Lumbar Vertebrae; D042282:Microdissection / Q000379:methods*; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis; D012720:Severity of Illness Index; D013122:Spinal Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology
D012333:RNA, Messenger