Modulation of the Inflammatory Response by Pre-emptive Administration of IMT504 Reduces Postoperative Pain in Rats and has Opioid-Sparing Effects.

Despite the available knowledge on underlying mechanisms and the development of several therapeutic strategies, optimal management of postoperative pain remains challenging. This preclinical study hypothesizes that, by promoting an anti-inflammatory scenario, pre-emptive administration of IMT504, a noncoding, non-CpG oligodeoxynucleotide with immune modulating properties, will reduce postincisional pain, also facilitating therapeutic opioid-sparing. Male adult Sprague-Dawley rats with unilateral hindpaw skin-muscle incision received pre-emptive (48 and 24 hours prior to surgery) or postoperative (6 hours after surgery) subcutaneous vehicle (saline) or IMT504. Various groups of rats were prepared for pain-like behavior analyses, including subgroups receiving morphine or naloxone, as well as for flow-cytometry or quantitative RT-PCR analyses of the spleen and hindpaws (for analysis of inflammatory phenotype). Compared to vehicle-treated rats, pre-emptive IMT504 significantly reduced mechanical allodynia by 6 hours after surgery, and accelerated recovery of basal responses from 72 hours after surgery and onwards. Cold allodynia was also reduced by IMT504. Postoperative administration of IMT504 resulted in similar positive effects on pain-like behavior. In IMT504-treated rats, 3 mg/kg morphine resulted in comparable blockade of mechanical allodynia as observed in vehicle-treated rats receiving 10 mg/kg morphine. IMT504 significantly increased hindpaw infiltration of mesenchymal stem cells, CD4+T and B cells, and caused upregulated or downregulated transcript expressions of interleukin-10 and interleukin-1ß, respectively. Also, IMT504 treatment targeted the spleen, with upregulated or downregulated transcript expressions, 6 hours after incision, of interleukin-10 and interleukin-1ß, respectively. Altogether, pre-emptive or postoperative IMT504 provides protection against postincisional pain, through participation of significant immunomodulatory actions, and exhibiting opioid-sparing effects. PERSPECTIVE: This preclinical study introduces the noncoding non-CpG oligodeoxynucleotide IMT504 as a novel modulator of postoperative pain and underlying inflammatory events. The opioid-sparing effects observed for IMT504 appear as a key feature that could contribute, in the future, to reducing opioid-related adverse events in patients undergoing surgical intervention.

IMT504 blocks allodynia in rats with spared nerve injury by promoting the migration of mesenchymal stem cells and by favoring an anti-inflammatory milieu at the injured nerve.

ABSTRACT: IMT504, a noncoding, non-CpG oligodeoxynucleotide, modulates pain-like behavior in rats undergoing peripheral nerve injury, through mechanisms that remain poorly characterized. Here, we chose the spared nerve injury model in rats to analyze the contribution of mesenchymal stem cells (MSCs) in the mechanisms of action of IMT504. We show that a single subcutaneous administration of IMT504 reverses mechanical and cold allodynia for at least 5 weeks posttreatment. This event correlated with long-lasting increases in the percentage of MSCs in peripheral blood and injured sciatic nerves, in a process seemingly influenced by modifications in the CXCL12-CXCR4 axis. Also, injured nerves presented with reduced tumor necrosis factor-α and interleukin-1ß and increased transforming growth factor-ß1 and interleukin-10 protein levels. In vitro analysis of IMT504-pretreated rat or human MSCs revealed internalized oligodeoxynucleotide and confirmed its promigratory effects. Moreover, IMT504-pretreatment induced transcript expression of Tgf-ß1 and Il-10 in MSCs; the increase in Il-10 becoming more robust after exposure to injured nerves. Ex vivo exposure of injured nerves to IMT504-pretreated MSCs confirmed the proinflammatory to anti-inflammatory switch observed in vivo. Interestingly, the sole exposure of injured nerves to IMT504 also resulted in downregulated Tnf-α and Il-1ß transcripts. Altogether, we reveal for the first time a direct association between the antiallodynic actions of IMT504, its promigratory and cytokine secretion modulating effects on MSCs, and further anti-inflammatory actions at injured nerves. The recapitulation of key outcomes in human MSCs supports the translational potential of IMT504 as a novel treatment for neuropathic pain with a unique mechanism of action involving the regulation of neuroimmune interactions.

IMT504 Provides Analgesia by Modulating Cell Infiltrate and Inflammatory Milieu in a Chronic Pain Model.

IMT504 is a non-CPG, non-coding synthetic oligodeoxinucleotide (ODN) with immunomodulatory properties and a novel inhibitory role in pain transmission, exerting long-lasting analgesic effects upon multiple systemic administrations. However, its mechanisms of anti-nociceptive action are still poorly understood. In the present study in male adult rats undergoing complete Freund's adjuvant-induced hindpaw inflammation, we focused in the analysis of the immunomodulatory role of IMT504 over the cellular infiltrate, the impact on the inflammatory milieu, and the correlation with its anti-allodynic role. By means of behavioral analysis, we determined that a single subcutaneous administration of 6 mg/kg of IMT504 is sufficient to exert a 6-week-long full reversal of mechanical and cold allodynia, compromising neither acute pain perception nor locomotor activity. Importantly, we found that the anti-nociceptive effects of systemic IMT504, plus quick reductions in hindpaw edema, were associated with a modulatory action upon cellular infiltrate of B-cells, macrophages and CD8+ T-cells populations. Accordingly, we observed a profound downregulation of several inflammatory leukocyte adhesion proteins, chemokines and cytokines, as well as of ß-endorphin and an increase in the anti-inflammatory cytokine, interleukin-10. Altogether, we demonstrate that at least part of the anti-nociceptive actions of IMT504 relate to the modulation of the peripheral immune system at the site of injury, favoring a switch from pro- to anti-inflammatory conditions, and provide further support to its use against chronic inflammatory pain. Graphical abstract GA short description - IMT504 systemic Administration. Systemic administration of the non-CpG ODN IMT504 results in a 6-week long blockade of pain-like behavior in association with anti-inflammatory responses at the site of injury. These include modulation of lymphoid and myeloid populations plus downregulated expression levels of multiple pro-inflammatory cytokines and ß-endorphin. Nocifensive responses and locomotion remain unaltered.

Transcript Expression of Vesicular Glutamate Transporters in Rat Dorsal Root Ganglion and Spinal Cord Neurons: Impact of Spinal Blockade during Hindpaw Inflammation.

Studies in mouse, and to a lesser extent in rat, have revealed the neuroanatomical distribution of vesicular glutamate transporters (VGLUTs) and begun exposing the critical role of VGLUT2 and VGLUT3 in pain transmission. In the present study in rat, we used specific riboprobes to characterize the transcript expression of all three VGLUTs in lumbar dorsal root ganglia (DRGs) and in the thoracolumbar, lumbar, and sacral spinal cord. We show for the first time in rat a very discrete VGLUT3 expression in DRGs and in deep layers of the dorsal horn. We confirm the abundant expression of VGLUT2, in both DRGs and the spinal cord, including presumable motorneurons in the latter. As expected, VGLUT1 was present in many DRG neuron profiles, and in the spinal cord it was mostly localized to neurons in the dorsal nucleus of Clarke. In rats with a 10 day long hindpaw inflammation, increased spinal expression of VGLUT2 transcript was detected by qRT-PCR, and intrathecal administration of the nonselective VGLUT inhibitor Chicago Sky Blue 6B resulted in reduced mechanical and thermal allodynia for up to 24 h. In conclusion, our results provide a collective characterization of VGLUTs in rat DRGs and the spinal cord, demonstrate increased spinal expression of VGLUT2 during chronic peripheral inflammation, and support the use of spinal VGLUT blockade as a strategy for attenuating inflammatory pain.

Long-lasting ameliorating effects of the oligodeoxynucleotide IMT504 on mechanical allodynia and hindpaw edema in rats with chronic hindpaw inflammation.

PURPOSE: Previously we showed that systemic administration of IMT504 prevents or ameliorates mechanical and thermal allodynia in rats with sciatic nerve crush. Here we analyzed if IMT504 is also effective in reducing mechanical allodynia and inflammation in rats undergoing hindpaw inflammation. MATERIALS AND METHODS: Male Sprague-Dawley rats received unilateral intraplantar injection of complete Freund́s adjuvant (CFA), and were grouped into: 1) untreated CFA, 2) vehicle-treated CFA, 3) IMT504-treated CFA (5 daily (5*) doses of 20, 2 or 0.2 mg/kg, or 3*2 mg/kg). Naïve groups were also included. Finally, early (immediately after intraplantar CFA) and late (7 days after intraplantar CFA) IMT504 treatment protocols were also tested. Hindpaw mechanical allodynia, dorsoventral thickness, edema and cellular infiltration of ipsilateral hindpaws were evaluated in all groups. RESULTS: Untreated CFA rats exhibited mechanical allodynia of quick onset (day 1) and long duration (7 weeks inclusive). Early and late treatments with 5*20 mg/kg IMT504 to CFA rats resulted in both quick and long-lasting antiallodynic effects, as compared to untreated CFA rats. This was also the case in CFA rats undergoing late IMT504 treatment at lower doses (3* and 5*2 mg/kg). Very low doses of IMT504 (5*0.2 mg/kg) only showed a mild improvement in withdrawal threshold, never reaching basal levels. Finally, rats treated with 3* or 5*2 mg/kg or 5*0.2 mg/kg exhibited significant decreases in dorsoventral thickness, edema, and inflammatory cell infiltration of the inflamed hindpaw. CONCLUSION: Early and late administration of IMT504 results in quick and long-lasting reductions in mechanical allodynia and hindpaw edema. While the mechanisms behind these effects remain to be established, data suggests that IMT504 administration could be a promising strategy in the control of inflammatory pain.

Spinal activation of the NPY Y1 receptor reduces mechanical and cold allodynia in rats with chronic constriction injury.

Neuropeptide tyrosine (NPY) and its associated receptors Y1R and Y2R have been previously implicated in the spinal modulation of neuropathic pain induced by total or partial sectioning of the sciatic nerve. However, their role in chronic constrictive injuries of the sciatic nerve has not yet been described. In the present study, we analyzed the consequences of pharmacological activation of spinal Y1R, by using the specific Y1R agonist Leu31Pro34-NPY, in rats with chronic constriction injury (CCI). CCI and sham-injury rats were implanted with a permanent intrathecal catheter (at day 7 after injury), and their response to the administration of different doses (2.5, 5, 7, 10 or 20µg) of Leu31Pro34-NPY (at a volume of 10µl) through the implanted catheter, recorded 14days after injury. Mechanical allodynia was tested by means of the up-and-down method, using von Frey filaments. Cold allodynia was tested by application of an acetone drop to the affected hindpaw. Intrathecal Leu31Pro34-NPY induced an increase of mechanical thresholds in rats with CCI, starting at doses of 5µg and becoming stronger with higher doses. Intrathecal Leu31Pro34 also resulted in reductions in the frequency of withdrawal to cold stimuli, although the effect was somewhat more moderate and mostly observed for doses of 7µg and higher. We thus show that spinal activation of the Y1R is able to reduce neuropathic pain due to a chronic constrictive injury and, together with other studies, support the use of a spinal Y1R agonist as a therapeutic agent against chronic pain induced by peripheral neuropathy.