ASICs and Surgical Pain

Dr. Eric Lingueglia, an INSERM group leader, and his team at the CNRS IPMC/IN2M have been doing impressive research using our i-Fect ™ siRNA Transfection Kits to study the role of Acid-Sensing Ion Channels in Postoperative Pain.

The etiology and pathophysiology of this pain is poorly understood. Their work is shedding light on potential root causes:
Emmanuel Deval, Jacques Noël, Xavier Gasull1, Anne Delaunay, Abdelkrim Alloui, Valérie Friend, Alain Eschalier, Michel Lazdunski, and Eric Lingueglia. Acid-Sensing Ion Channels in Postoperative Pain. The Journal of Neuroscience, 20 April 2011, 31(16): 6059-6066; doi: 10.1523/​JNEUROSCI.5266-10.2011.

...Ten microliters of a siRNA (2 μg)/i-Fect (Neuromics) mix was injected intrathecally between the L4 and L5 vertebrae of rats using a Hamilton syringe and a 25 gauge needle. Animals received one injection per day for 4 d (Fig. 4A, protocol). ASIC3 (CUACACGCUAUGCCAAGGAdtdt) and the corresponding scramble (GCUCACACUACGCAGAGAUdtdt) siRNAs have been previously described (Deval et al., 2008)...


Highlights: Pharmacological inhibition of ASIC3 channels with the specific toxin APETx2 or in vivo knockdown of ASIC3 subunit by small interfering RNA led to a significant reduction of postoperative spontaneous, thermal, and postural pain behaviors (spontaneous flinching, heat hyperalgesia, and weight bearing). ASIC3 appears to have an important role in deep tissue but also affects prolonged pain evoked by skin incision alone.

ASIC3s are excitatory ion channels directly activated by extracellular protons that detect the painful drops in pH at incision points. Several factors may participate in the drop of extracellular pH, such as release of the acidic content of lyzed cells, degranulation of mast cells, organic acids released by metabolism..etc  This makes makes the Ion Channel a great marker for the studying activation of pain and a potential therapeutic target for mitigating surgical pain.

I will continue to track and report progress.

i-Fect and siRNA Delvery to Toll-like receptor 4

I have reported use of our i-Fect^TM siRNA delivery kit for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, Survivin,  Flaviviruses and more.

These represent potential targets for Pain, Cancer and Infectious Disease Therapies.

The latest study involves successful knockdown of the Toll-like receptor 4 (TLR-4):

Wu Fx, Bian Jj, Miao Xr, Huang Sd, Xu Xw, Gong Dj, Sun Ym, Lu Zj, Yu Wf. Intrathecal siRNA against Toll-like receptor 4 reduces nociception in a rat model of neuropathic pain. Int J Med Sci 2010; 7:251-259.

Background: Neuropathic pain is characterized by hyperalgesia, allodynia and spontaneous pain. It often occurs as a result of injury to peripheral nerves, dorsal root ganglions (DRG), spinal cord, or brain. Recent studies have suggested that Toll-like receptor 4 (TLR4) might play a role in neuropathic pain. Methodology/Principal Findings: In this study, we investigated the role of TLR4 in a rat chronic constriction injury (CCI) model and explored the feasibility of treating neuropathic pain by inhibiting TLR4. Our results demonstrated that intrathecal siRNA-mediated suppression of TLR4 attenuated CCI-induced mechanical allodynia and thermal hyperalgesia through inhibiting the activation of NF-κB p65 and production of proinflammatory cytokines (e.g., TNF-α and IL-1β). Conclusions/Significance: These findings suggest that suppression of TLR4 mediated by intrathecally administered siRNA may be a new strategy for the treatment of neuropathic pain.

Images: Screening siRNA for an efficient suppression of TLR4 expression in vitro. HEK-293 cells were co-transfected with both pEGFRC1-TLR4 and either one of three independent siRNA oligonucleotides targeting TLR4 (TLR4-siRNA1-3) or a control siRNA (MM-siRNA). Two days after transfection, EGFP fluorescence was observed under microscope (A) or quantified by flow cytometry (B). (A) EGFP fluorescence under an inverted fluorescence microscope (×100) or cell density under an optical microscope (×100). A, control; B, siRNA1; C, siRNA2; D, siRNA3. (B) The quantification of TLR4-EGFP fluorescence intensity upon siRNA knockdown was evaluated by flow cytometry analysis. Immunofluorescence and flow cytometry results revealed that all 3 siRNAs had efficient inhibition on GFP fluorescence, and TLR4-siRNA2 was the most potent.

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.