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!

i-Fect, Survivin and Gliobastomas

I would like to add Survivin to the list of genes successfully silenced in-vitro and in-vivo using our i-Fect^TMsiRNA delivery kit.

The list includes: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more

Joseph George, Naren L. Banik and Swapan K. Ray. Survivin knockdown and concurrent 4-HPR treatment controlled human glioblastoma in vitro and in vivo. Neuro-Oncology, doi:10.1093/neuonc/noq079.

...survivin siRNA cDNA was suspended in RNAse free sterile water (25 μg DNA/10 μl) and mixed (1:4 v/v) with i-Fect transfection reagent (Neuromics)...

Delivery of the Surivivin siRNA resulted in significant decreases in Glioblatoma Tumor Size.

Direct Application of siRNA for In Vivo Pain Research

My friends at McGill University have recently published in depth methods for using siRNA to study pain. Dr. Philippe Sarret have done extensive work delivering siRNA + i-Fect^TM in vivo for gene expression analysis of specific pain receptors.

Here's a link to the book chapter from Springer Protocols:

25. Direct Application of siRNA for In Vivo Pain Research
By: Philippe Sarret , Louis Doré-Savard, Nicolas Beaudet
Affiliation(s): (1) Department of Physiology and Biophysics, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada
Book Title: RNA Interference: From Biology to Clinical Applications
Series: Methods in Molecular Biology Volume: 623 Pub. Date: May-01-2010 Page Range: 383-395 DOI: 10.1007/978-1-60761-588-0_25

Abstract: Pain is the new burden of the twenty-first century, raising enormous socio-economic costs to developed and underdeveloped countries. Chronic pain is a central nervous system (CNS) pathology, affecting a large proportion of the population. Morphine and its derivatives are still the golden clinical standards for treating pain although they induce severe side effects. To this day, we still have poor understanding of nociceptive pain and its underlying complex mechanisms; furthermore, novelty in clinical analgesics is lacking.

RNA interference technologies are promising both for pain research and treatment. This genetic approach will likely provide new insights into pain mechanisms and eventually offer nonpharmacological therapeutic approaches. In vivo research is thus crucial to reach this goal. Preclinical studies on rodents are necessary to validate small interfering RNA (siRNA) candidates and to target precise physiological pain modulators. Aiming treatment at the CNS is delicate work, and here we will describe how to perform adequate pain research using siRNA, including siRNA preparation and injection, animal behavioral models, and CNS tissue collection.

Knockdown of rSNSR1 in vivo

The parade of success with use our i-FectTM in vivo grows. Here's the most recent study:

Christian Ndong, Amynah Pradhan, Carole Puma, Jean-Pierre Morello, Cyrla Hoffert, Thierry Groblewski , Dajan O’Donnell, Jennifer M.A. Laird. Role of rat sensory neuron-specific receptor (rSNSR1) in inflammatory pain: Contribution of TRPV1 to SNSR signaling in the pain pathway. PAIN 143 (2009) 130–137.
...For experiments in which siRNA was delivered by bolus injections, 10 ul of siRNA or vehicle was injected directly into the intrathecal catheter once daily for 4 days. In this case, siRNAs were prepared immediately prior to administration by mixing the RNA solution (200 uM in annealing buffer) with the transfection reagent i-FectTM (Neuromics) at a ratio of 1:4 (w:v) for a final siRNA/ lipid complex concentration of 2 ug/10 ul...

Related Data:



Images: in vivo characterization of knockdown produced by rSNSR1 siRNA. (A) A dose-dependent decrease in rSNSR1 mRNA levels measured in lumbar L3/L4/L5 DRGs was
observed when rSNSR1 siRNA (n = 7–14/group) or MM siRNA (n = 6/group) was delivered by four daily bolus injections. *p < 0.05; **p < 0.01; ***p < 0.001 as determined by oneway analysis of variance followed by sequential testing. (B) rSNSR1 immunoreactivity in dorsal horn of the spinal cord was visibly reduced in rSNSR1 siRNA-treated animals (5 lg/day, left panel). Immunoreactivity with neuron-specific isolectin B4 (IB4; right panel) did not change between treatment groups, showing the integrity of each dorsal horn analyzed (n = 6/group). (C) A semi-quantitative score of rSNSR1 immunoreactivity showed that siRNA treatment greatly decreased rSNSR1 protein levels compared to MM and control groups. A blinded observer scored 9–12 individual sections taken from a 1 cm segment of the spinal cord.

Intrathecal Delivery of siRNA

We wanted to present yet another publication referencing successful delivery of siRNA using i-Fect^TM:

Suneeta Tumati, Tally Largent Milnes, Henry I. Yamamura, Todd W. Vanderah, William R. Roeske and Eva V. Varga. Intrathecal Raf-1-selective siRNA attenuates sustained morphine-mediated thermal hyperalgesia. doi:10.1016/j.ejphar.2008.10.033

...siRNAs stock solutions (100 μM) were prepared in double distilled RNAse free water and stored in aliquots at −80 °C. For intrathecal treatment, aliquots of the stock solution (2 μg of the appropriate siRNA) were mixed (1:5 v/v)with i-Fect transfection reagent (Neuromics, Edina, MN). After recovery from the surgery (5–7 days), the animals received intrathecal injections (2 ug siRNA/10 ul/rat) of either a lipid encapsulated Raf-1-selective siRNA mixture (Smart pool siRNA, Dharmacon Inc; Chicago, IL, Cat # L-087699-00) (Raf-1 siRNA groups) or i-Fect encapsulated non-targeting dsRNA (Dharmacon, #D-001810-01-20) (control mismatch siRNA groups) or the transfection lipid alone, once daily, for 3 days, as described earlier (Gardell et al., 2002). Intrathecal injections of the siRNAs or the transfection agent alone did not cause any sign of behavioral toxicity. Western blots, using a Raf-1-selective antibody, indicated that intrathecal treatment with the Raf-1-selective siRNA mixture for 3 days significantly reduced Raf-1 protein levels in the dorsal root ganglion and in the dorsal horn of the spinal cord...