Evaluating the Analgesic Effect of the GLS Inhibitor 6-Diazo-5-Oxo-L-Norleucine in Vivo.

Glutamate is an excitatory neurotransmitter, produced by its synthetic enzyme, glutaminase (GLS), and packaged by vesicular transporters (VGluT2) into synaptic vesicles. Primary sensory peripheral nerve and spinal synaptic terminals release glutamate during nociceptive (pain) signaling. In post-incisional and inflammation models in rats, GLS and VGluT2 production is elevated in dorsal root ganglion neuronal cell bodies and transported to peripheral and spinal terminals for increased glutamate synthesis and release. 6-Diazo-5-oxo-l-norleucine (DON) is a GLS inhibitor that produces long lasting pain relief when applied to the inflamed paw of arthritic rats, but its effect in a post-incisional model has not been evaluated. In this study, we examined the analgesic efficacy of DON in a surgical incision model by measuring thermal latency and mechanical allodynia. Following behavioral evaluation, we examined the skin for VGluT2, GLS and glutamate immunoreactivity (ir). Our findings revealed that VGluT2-ir is elevated in the stratum lucidum by approximately 19%, 64 hours post-surgical incision and attenuated by approximately 5.4% after the administration of DON. During that same period GLS-ir was elevated in dermal nerve fibers by 52% and was attenuated by approximately 27.9% after the application of DON. Additionally, glutamate-ir was elevated in epidermal nerve fibers by 35% after incision and attenuated by approximately 23% after the administration of DON. Behavioral testing 24 and 48 hours after a single local administration of DON showed five out of six animals having an analgesic response to mechanical allodynia, but not to thermal hyperalgesia. Following a surgical incision, the area of injury shows increased VGluT2-, GLS-, glutamate-ir, mechanical allodynia and no change in thermal latency. After the application of the GLS inhibitor, DON, nerve fiber of the skin showed decreased VGluT2, GLS, and glutamate-ir. Furthermore, post-incision DON treated animals exhibited decreased mechanical allodynia with no change in thermal latency when compared to control animals.