Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model.
BACKGROUND
The etiology of degenerative disc disease is unknown. Several investigators have reported the presence of proteolytic enzymes, such as the matrix metalloproteinase (MMP) and ADAMTS (a disintegrin and metalloprotease with thrombospondin-like repeats) families, in degenerated human discs. Glasson and colleagues recently reported that a significant reduction occurs in the severity of cartilage destruction in ADAMTS5 knockout mice compared with wild-type mice. The purpose of this study was to evaluate the suppressive effects of injections of ADAMTS5 small interference RNA (siRNA) oligonucleotide on intervertebral disc degeneration in the rabbit anular needle-puncture model.


METHODS
Rabbit nucleus pulposus (NP) cells were transfected with siRNA oligonucleotides specific for ADAMTS5 or the control. The suppression of the ADAMTS5 gene by siRNA transfection was assessed by using real-time polymerase chain reaction (PCR), both in monolayer and alginate bead cultures with or without interleukin-1beta (IL-1beta) stimulation. The effect of siRNA was determined in vivo by using the rabbit anular needle-puncture model (control group: n = 8; ADAMTS5 group: n = 8). One week after the initial anular puncture, the animals received an injection of the control or anti-ADAMTS5 oligonucleotide (100 microg each at the L2/3 and L4/5 level; 16 discs/group). Disc height, magnetic resonance imaging (MRI) (Thompson classification and signal intensity), and safranin-O staining (histologic grade) were assessed.


RESULTS
IL-1beta treatment significantly increased the ADAMTS5 mRNA level in NP cells (P < 0.01). ADAMTS5 gene suppression was 70% compared with the control oligonucleotide in both monolayer and alginate bead culture with or without stimulation with IL-1beta. The injection of anti-ADAMTS5 oligonucleotide in vivo resulted in improved MRI scores with increased signal intensity and improved histologic grade scores with statistical significance (P < 0.05). No significant change in disc height was observed.


CONCLUSIONS
A single injection of ADAMTS5 siRNA induced the suppression of degradation in NP tissues, as shown by significantly improved MRI and histologic grades. The mechanism of response to siRNA may be worthy of exploration for possible therapeutic purposes.
Seki
Shoji
S
Department of Orthopaedic Surgery, Faculty of Medicine, University of Toyama, 2630 Sugitani, Toyama, Toyama 930-0194, Japan. seki@med.u-toyama.ac.jp
Asanuma-Abe
Yumiko
Y
Masuda
Koichi
K
Kawaguchi
Yoshiharu
Y
Asanuma
Kunihiro
K
Muehleman
Carol
C
Iwai
Akiko
A
Kimura
Tomoatsu
T
D051722:ADAM Proteins / Q000378:metabolism*; D000818:Animals; D007091:Image Processing, Computer-Assisted; D055959:Intervertebral Disc Degeneration / Q000201:enzymology* / Q000473:pathology; D008279:Magnetic Resonance Imaging; D034741:RNA, Small Interfering; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D034741:RNA, Small Interfering; D051722:ADAM Proteins