Long-lasting antiallodynic effects of IMT504 in rats with spared nerve injury relate to strengthened migration of mesenchymal stem cells towards injured nerves

M. CASADEI; L. DOMINGUEZ; M. GARCÍA; M.J. VILLAR; J. RUBIONE; M.F. CORONEL; G. MAZZOLINI; P.R. BRUMOVSKY; E. FIORE; C. LEIGUARDA; H.TRIGOSSO-VENARIO; A.D. MONTANER

Congreso; XXXV Reunión Anual de la Sociedad Argentina de Investigación en Neurociencias; 2020

Sociedad Argentina de Investigación en Neurociencias

Background and Aims: Peripheral neuropathic pain is caused by injury or dysfunction of peripheral nerves, and is characterized by allodynia and hyperalgesia. Neuropathic pain is complex and difficult to treat, in many cases resistant to currently available pain drugs. We have shown that multiple systemic doses of the oligodeoxynucleotide (ODN) IMT504 result in clear and long-lasting antiallodynic and anti-inflammatory effects in rats with unilateral sciatic nerve crush or hindpaw inflammation. Interestingly, in rats with sciatic nerve crush, virtually identical allodynia-preventing effects were observed after systemic administration of IMT504 or exogenous rat bone marrow mesenchymal stem cells (BMMSC). Here, we address the role of IMT504 in a model of chronic neuropathic pain, and the involvement of BMMSC. Methods: All procedures were performed on adult male Sprague-Dawley rats.The spared nerve injury (SNI) model was used to induce persistent neuropathic pain. Subcutaneous administration of IMT504 (6mg/kg) was used. Two different protocols were tested: 1) Early treatment (ET; IMT504 administration on the same day of SNI), and 2) late treatment (LT; IMT504 administration 7 days after SNI). In all cases, control animals received a subcutaneous injection of sterile saline (Vehicle-treated rats). Mechanical and cold allodynia were measured in contra- and ipsilateral hindpaws, before and for several days and weeks after treatment. BMMSC migration was addressed in vivo in rats under the IMT504-LT protocol, by means of flow cytometry analysis (for identification of specific antigens; CD45-, CD90+, CD29+). Bone marrow, peripheral blood and sciatic nerves at different survival times (10 and 28 days after SNI) were used. In vitro migration assays were performed to determine if IMT504 pre-treatment (7 g/ml; overnight (ON)) promotes the migration of BMMSCs towards sciatic nerve conditioned medium (SNCM). Contra- and ipsilateral SNCM were obtained by ON culturing of nerves dissected from naïve rats or 7-day SNI rats. Rat BMMSCs were collected from femur and tibia. Human BMMSCs were obtained from healthy donors (Hospital Naval Pedro Mallo, Buenos Aires, Argentina). All cells were cultured until passage 4 was achieved. In vitro migration was performed using a Multiwell Chemotaxis Chamber (Neuroprobe, Inc.). After 5 hours at 37°C in a 5% CO2 humidified atmosphere, cells attached to the membrane were fixed and stained with 4?,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) and counted using fluorescent-field microscopy. Results: All SNI rats exhibited marked increases in ipsilateral mechanical and cold allodynia, reaching allodynic levels as early as 3 days after injury. Vehicle-treated injured rats remained allodynic throughout the entire tested period (6 weeks). In contrast, ET and LT with IMT504 resulted in clear antiallodynic signs in injured rats. Animals receiving IMT504-ET showed a tendency towards reduced mechanical and cold allodynia 3 days after injury, this reduction becoming clear afterwards. Rats receiving IMT504-LT showed clear reductions in mechanical and cold allodynia already 1 day after treatment, the effect becoming stronger in the days and weeks after. In both treatment protocols, rats remained non-allodynic up to the 6 weeks tested. The impact of IMT504-LT on BMMSCs mobilization, migration and homing was tested 3 and 21 days after treatment. In injured rats receiving vehicle, a large number of BMMSCs were detected in peripheral blood and injured sciatic nerves, as compared to naïve rats. In contrast, IMT504-treated SNI rats always showed the greatest number of BMMSCs at both sites, compared to the other two experimental groups. Contralateral nerves did not show significant homing of BMMSCs, as addressed 21 days after vehicle or IMT504 treatment, and compared to naïve nerves. Analysis in bone marrow showed that injured rats receiving vehicle or IMT504-LT exhibit reductions in BMMSCs. Finally, in vitro migration assays showed that IMT504 potentiates the capacity of rat and human BMMSCs to migrate towards SNCM, the effect being stronger for medium obtained from 7-day-injured sciatic nerves. Conclusions: Early or late IMT504 administration revert mechanical and cold allodynia in animals undergoing persistent neuropathic pain. The effect exhibits a considerably quick onset and is long-lasting. The ODN also appears to potentiate the mobilization, migration and homing of BMMSCs into injured nerves. If these effects on BMMSCs relate to the antiallodynic actions of IMT504, it remains to be further demonstrated. However, our results support the idea that this ODN could be a promising therapeutic agent in the treatment of chronic neuropathic pain, also in humans. Financial Support: PICTO-startup (2016-0091), PICT (2017-0969), IBRO, Fondecyt (1181622), CEDENNA FB 0807. Ethical Permissions: IACUC-IIMT-17-02.