SiRNA-Mediated Knockdown of the NR1 Subunit Gene of the NMDA Receptor Attenuates Formalin-Induced Pain Behaviors in Adult Rats

Abstract

NMDA receptors in the spinal cord dorsal horn (SCDH) mediate some inflammatory pain behaviors. Here, we used rAAV vectors expressing an active small interfering RNA (siRNA) (vector 6) targeting the essential NR1 subunit of the NMDA receptor or a mismatch siRNA (vector MM-6) sequence to determine the consequences of RNAi-mediated knockdown of NR1 expression on NMDA receptor levels and formalin-induced pain behaviors in adult rats. Three weeks after intraparenchymal administration of the vector 6 into the right lumbar SCDH, NR1 mRNA and protein levels were significantly reduced (P < .01) in the ipsilateral SCDH compared with the contralateral SCDH but not in vector MM-6 or non-vector control animals. Formalin-induced phase 2 nociceptive response was significantly reduced (P < .05) in vector 6 animals compared with controls. Although neither vector affected normal mechanical threshold, vector 6 provided protection from the mechanical allodynia seen in controls at 24 hours after intraplantar formalin. Vector 6 also prevented the increase in phosphorylated NR1 levels seen in the ipsilateral SCDH of control rats 45 minutes after formalin. These results indicate that vector-derived siRNAs can effectively produce spatial knockdown of NR1 gene expression, and this knockdown selectively attenuates in vivo NMDA receptor-mediated formalin behaviors and NR1 phosphorylation in the rat. This study reveals that a single administration of an siRNA-expressing viral vector produces significant knockdown of the NR1 gene in the SCDH of adult rats. This preclinical study demonstrates the use of RNAi to target the expression of genes mediating pain and the therapeutic potential of this approach.