Purinergic receptor antagonist modulates hyperalgesia and allodynia in a mouse model of neuropathy : involvement of NO system at central and peripheral nervous system level

Neuropathic pain, consequent to peripheral injury, has been associated with upregulation of NOS expression and subsequent NO overproduction at level of both injured sciatic nerve and spinal cord. Animal models of nerve injury provide a reliable tool for investigating the mechanisms underlying neuropathic pain. First aim of this work was to study NO and its associated biosynthetic enzymes, at different pain transmission anatomic steps, in mononeuropathy induced by chronic constriction injury of C57BL/65 mice sciatic nerve. The paw withdrawal latency and the sensitivity to tactile stimuli were evaluated at 3, 7 and 14 days after injury by the plantar test and the dynamic plantar aesthesiometer, respectively. At the same times, thalamus, L4-L6 dorsal spinal cord, corresponding ipsilateral dorsal root ganglia (DRGs) and ipsilateral sciatic nerve, proximal to injury, were removed using a dissecting microscope. Neuronal and inducible nitric oxide synthase (i and nNOS) content was measured by western immunoblotting. Nitric oxide production was assessed on the basis of nitrite/nitrate, which are the oxidation end products of nitric oxide, using a fluorimetric procedure. Hyperalgesia and allodynia were already maximal at 3 days and they remained elevated up to 14 days. On 3rd day, the expression of iNOS was unmodified both locally and centrally and only at subsequent evaluation times increased in central and peripheral nervous system. In contrast, nNOS was precociously elevated in contralateral thalamus and only later it was upregulated in the peripheral nervous tissues. Nitric oxide content increased in all tissues, except for contralateral thalamus, at 7 and 14th day. These results support the hypothesis that NO-NOS system is involved in neuropathic pain, particularly nNOS seems to have a role on its development and maintenance; instead iNOS in later phases. So it appears to be important marker of painful neuropathy both in the central and peripheral nervous system. ATP activates cation ion channels, P2X receptors, and G-protein-coupled P2Y receptors and it is now recognised as an endogenous mediator of pain. The second objective was to evaluate the possible role of purinergic signalling in hyperalgesic and allodynic responses. Therefore we determined the effects of a non-selective P2 purinergic receptor-antagonist, pyridoxalphosphate- 6-azophenyl-2’,4’-disulfonic acid (PPADS), on pain behaviour and increase in NONOS system. The administration in mice of PPADS (6.25, 12.5 and 25 mg/kg, i.p.), once a day for eleven days, from day 3 after the nerve injury, dose and time dependently attenuated both mechanical allodynia and hyperalgesia. The complete antiallodynic and antihyperalgesic efficacy of PPADS 25 mg/kg was associated with 1) reduction in nNOS content of contralateral thalamus, DRGs and sciatic nerve, 2) decrease in overexpressed iNOS in all tissues and, consequently, 3) inhibition of NO overproduction. Finally, we can hypothesize that PPADS is therapeutically effective, because it binds the different subtypes of purinoceptors (P2X and P2Y) involved in neuropathic pain.