Pannexin 1 (panx1) is a large-pore membrane channel expressed in many tissues of mammals, including neurons and glial cells. Panx1 channels are highly permeable to calcium and adenosine triphosphatase (ATP); on the other hand, they can be opened by ATP and glutamate, two crucial molecules for acute and chronic pain signaling in the spinal cord dorsal horn, thus suggesting that panx1 could be a key component for the generation of central sensitization during persistent pain. In this study, we examined the effect of three panx1 blockers, namely, 10panx peptide, carbenoxolone, and probenecid, on C-reflex wind-up activity and mechanical nociceptive behavior in a spared nerve injury neuropathic rat model involving sural nerve transection. In addition, the expression of panx1 protein in the dorsal horn of the ipsilateral lumbar spinal cord was measured in sural nerve-transected and sham-operated control rats. Sural nerve transection resulted in a lower threshold for C-reflex activation by electric stimulation of the injured hindpaw, together with persistent mechanical hypersensitivity to pressure stimuli applied to the paw. Intrathecal administration of the panx1 blockers significantly depressed the spinal C-reflex wind-up activity in both neuropathic and sham control rats, and decreased mechanical hyperalgesia in neuropathic rats without affecting the nociceptive threshold in sham animals. Western blotting showed that panx1 was similarly expressed in the dorsal horn of lumbar spinal cord from neuropathic and sham rats. The present results constitute the first evidence that panx1 channels play a significant role in the mechanisms underlying central sensitization in neuropathic pain.

Introduction: Cytokines produced by spinal cord glia after peripheral injuries have a relevant role in the maintenance of pain states. Thus, while IL-1β is overexpressed in the spinal cords of animals submitted to experimental arthritis and other chronic pain models, intrathecal administration of IL-1β to healthy animals induces hyperalgesia and allodynia and enhances wind-up activity in dorsal horn neurons.Methods: To investigate the functional contribution of glial cells in the spinal cord nociceptive transmission, the effect of intrathecally administered IL-1β was studied in both normal and adjuvant-induced arthritic rats with or without glial inhibition. Four weeks after induction of monoarthritis, rats were treated with the glial cell inhibitor propentofylline (10 μg i.t. daily during 10 days) and submitted to a C-fiber-mediated reflex paradigm evoked by single and repetitive (wind-up) electric stimulation.Results: Both the propentofylline treatment and the monoarthritic condition modified the stimulating current required for threshold activation of C reflex responses. Intrathecal IL-1β increased spinal cord wind-up activity in normal and monoarthritic rats without propentofylline pre-treatment, but resulted in decreased wind-up activity in normal and monoarthritic propentofylline-treated animals. Intrathecal saline did not produce any effect. Thus, glial inactivation reverted into inhibition the excitatory effect of IL-1β on spinal cord wind-up, irrespective of the normal or monoarthritic condition of rats.Conclusions: The results suggest that the excitatory effect of nanomolar doses of IL-1β on spinal wind-up in healthy rats is produced by an unidentified glial mediator, while the inhibitory effects of IL-1β on wind-up activity in animals with inactivated glia resulted from a direct effect of the cytokine on dorsal horn neurons. The present study failed to demonstrate a differential sensitivity of normal and monoarthritic rats to IL-1β administration into the spinal cord and to disruption of β glial function, as both normal and monoarthritic animals changes wind-up activity in the same direction after propentofylline treatment, suggesting that after glial inhibition normal and monoarthritic animals behave similarly relative to the capability of dorsal horn neurons to generate wind-up activity when repeatedly stimulated by C-fibers.

Pannexin 1 (Panx1) is involved in the spinal central sensitization process in rats with neuropathic pain, but its interaction with well-known, pain-related, ligand-dependent receptors, such as NMDA receptors (NMDAR) and P2X7 purinoceptors (P2X7R), remains largely unexplored. Here, we studied whether NMDAR- and P2X7R-dependent nociceptive signaling in neuropathic rats require the activation of Panx1 channels to generate spinal central sensitization, as assessed by behavioral (mechanical hyperalgesia) and electrophysiological (C-reflex wind-up potentiation) indexes. Administration of either a selective NMDAR agonist i.t. (NMDA, 2 mM) or a P2X7R agonist (BzATP, 150 μM) significantly increased both the mechanical hyperalgesia and the C-reflex wind-up potentiation, effects that were rapidly reversed (minutes) by i.t. administration of a selective pannexin 1 antagonist (10panx peptide, 300 μM), with the scores even reaching values of rats without neuropathy. Accordingly, 300 μM 10panx completely prevented the effects of NMDA and BzATP administered 1 h later, on mechanical hyperalgesia and C-reflex wind-up potentiation. Confocal immunofluorescence imaging revealed coexpression of Panx1 with NeuN protein in intrinsic dorsal horn neurons of neuropathic rats. The results indicate that both NMDAR- and P2X7R-mediated increases in mechanical hyperalgesia and C-reflex wind-up potentiation require neuronal Panx1 channel activation to initiate and maintain nociceptive signaling in neuropathic rats.

Drug delivery systems based on polymeric microparticles represent an interesting field of development for the treatment of several infectious diseases for humans and animals. In this work, we developed PLGA microparticles loaded with ceftiofur (PLGA-cef), a third- generation cephalosporin that is used exclusively used in animals. PLGA-cef was prepared by the double emulsion w/o/w method, and exhibited a diameter in the range of 1.5–2.2 μm, and a negative ζ potential in the range of -35 to -55 mV. The loading yield of PLGA-cef was ~7% and encapsulation efficiency was approximately 40%. The pharmacokinetic study demonstrated a sustained release profile of ceftiofur for 20 days. PLGA-cef administrated in a single dose was more effective than ceftiofur non-encapsulated in rats challenged with S. Typhimurium. The in vivo toxicological evaluation showed that PLGA-cef did not affect the blood biochemical, hematological and hemostasis parameters. Overall, the PLGA-cef showed slow in vivo release profile, high antibacterial efficacy, and low toxicity. The results obtained supports the safe application of PLGA-cef as sustained release platform in the veterinary industry.