Sunday, February 26, 2012

Silencing NOV in vivo using i-Fect

Implications for Treating Neuropathic or Inflammatory Pain

It has been a awhile since I posted results here for researchers using Neuromics' i-Fect ™ siRNA Transfection Kits. Over the past months, we have enjoyed more growth in use of these kits so I anticipate more positive results to come.

Inflammation plays and evil role in Neuropathic Pain. Sustained neuroinflammation cased by  release of pro-inflammatory cytokines and chemokines (including TNF-α, IL-1β, IL-6 and CCL2). Emerging studies have established that the extracellular matrix (ECM) components, particularly matrix metalloproteinases (MMPs) actively participate in the generation and maintenance of pain.
In this study, investigators show how modulating expression of nephroblastoma overexpressed gene (NOV) can mitigate expression of the MMPs and thus regulate Pain: Lara Kular, Cyril Rivat, Brigitte Lelongt, Claire Calmel, Maryvonne Laurent, Michel Pohl, Patrick Kitabgi, Stephane Melik-Parsadaniantz and Cecile Martinerie. NOV/CCN3 attenuates inflammatory pain through regulation of matrix metalloproteinases-2 and -9. Journal of Neuroinflammation 2012, 9:36 doi:10.1186/1742-2094-9-36.
Results:  NOV was expressed in neurons of both dorsal root ganglia (DRG) and dorsal horn of the spinal cord (DHSC). After intraplantar CFA injection, NOV levels were transiently and persistently down-regulated in the DRG and DHSC, respectively, occurring at the maintenance phase of pain (15 days). NOV-reduced expression was restored after treatment of CFA rats with dexamethasone. In vitro, results based on cultured DRG neurons showed that siRNA-mediated inhibition of NOV enhanced IL-1beta- and TNF-alpha-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-alpha-induced MMP-9 expression through beta1 integrin engagement. In vivo, the intrathecal delivery of MMP-9 inhibitor attenuated mechanical allodynia of CFA rats. Importantly, intrathecal administration of NOV siRNA specifically led to an up-regulation of MMP-9 in the DRG and MMP-2 in the DHSC concomitant with increased mechanical allodynia. Finally, NOV intrathecal treatment specifically abolished the induction of MMP-9 in the DRG and, MMP-9 and MMP-2 in the DHSC of CFA rats. This inhibitory effect on MMP is associated with reduced mechanical allodynia.
Conclusions:  This study identifies NOV as a new actor against inflammatory pain through regulation of MMPs thus uncovering NOV as an attractive candidate for therapeutic improvement in pain relief.

Figure 9. Effect of in vivo endogenous NOV inhibition on MMP-2/-9 expression and mechanical allodynia. In CFA rats, NOV-specific siRNA (2 μg) or control non-silencing siRNA (Ctr) were delivered intrathecally (i.t) daily for 3 consecutive days. (A) NOV protein levels in DHSC. Representative western blot (left panel) and quantification of protein levels normalized to GAPDH (right panel) (**P <0.01, siNOV versus Ctr, n = 6) (B, C) Levels of MMP-9 and MMP-2 mRNA in DRG (B) and DHSC. (C) Transcript levels were quantified by RT-qPCR and values were normalized to rat S26 mRNA level. Data represent the mean value ± SEM of two independent experiments realized with three rats per condition (*P <0.05 siNOV versus Ctr). (D) Representative gelatin zymograph showing MMP-9 and MMP-2 activities in DRG (left panel) and quantification of MMP-2 and MMP-9 gelatinolytic bands (right panel). Data represent the mean ± SEM of six rats per group (**P <0.01 siNOV versus Ctr). (E) Paw withdrawal threshold (g) of CFA rats intrathecally injected with NOV-specific siRNA or control siRNA evaluated using the von Frey test. Data represent the mean ± SEM of eight rats per group (*P <0.05 siNOV- versus Ctr-treated rats), BL: baseline In order to test whether endogenously produced NOV could modulate inflammatory pain, we evaluated the mechanical allodynia of CFA rats treated with NOV. As shown in Figure 9E, intrathecal delivery of siNOV resulted in a significant increase of mechanical allodynia compared to rats injected with control siRNA (*P <0.05, n = 8). These data strongly suggest that endogenously produced NOV influences pain intensity and further support the hypothesis that NOV downregulation could participate in pain processes through
upregulation of MMP-2 and MMP-9.

Learn more about Neuromics' Transfection Reagents!

No comments: