Composition Of INOS Immunoreactive Cells In The Spinal Dorsal Horn During Formalin Induced Inflammatory Pain

Objective: Nitric oxide (NO) is an important pain mediator, and NO synthase (NOS) is the rate-limiting enzyme in the NO production. Many reports showed that peripheral nociceptive stimulus could upregulate expression of NOS and NO production, and the up-regulated NOS and NO participated in the development and maintenance of pathological pain. Our previous studies using NADPH-d histochemistry and immuno- histochemistry showed that nNOS and iNOS expression in the dorsal horn of the spinal cord increased during formalin induced inflammatory pain, and further proved the important role of NO in the development and maintenance of pathological pain. However, the composition of the iNOS immunoreactive cells in the process is remained to be clarified.A variety of evidence indicated the association of spinal glia (microglia and astroglia) with the development and maintenance of pathological pain. For instance, reversible inhibitor of glia metabolism could inhibit the spontaneous pain and allodynia in many experimental pain models. Spinal glia could be activated in neuropathological or peripheral inflammatory pain models. The activated glia could release many substances such as interleyukin-1, tumor necrosis factors, NO etc to facilitate pain transmission. Therefore, besides neurons, glia may be another type of cell involved in the upregulation of iNOS expression during formalin induced pathological pain.The present study was undertaken to observe the contribution of spinal astrocytes and microglia in the upregulation of iNOS during formalin-induced pathological pain, and to further illustrate the important role of NO and spinal glia in peripheral inflammatory pain. For the purpose, double-labeled immunohistochemistry for iNOS and GFAP (specific protein for astrocyte) expression and comparison between iNOS and OX42(specific protein for microglia) expression using immunohistochemical staining in series sections were performed in the spinal dorsal horn of rats subjected to formalin inflammatory pain.Methods: Thirty male Spague-Dawley rats were divided randomly into normal saline (NS), and formalin groups. Rats in each group were subcutaneously injected with NS or formalin solution (5%) into plantar surface of the right hindpaw. According to the observed time after NS or formalin injection, each group was further divided into 1 h, 24 h and 48 h subgroups. The L5 segment of spinal cord was dissected out at the designed ending time point and sectioned in paraffin preparation for iNOS and GFAP double-labeled immuno- histochemistry staining.For comparing expression of iNOS with that of OX42 using immunohistochemical staining, another thirty male Spague-Dawley rats were divided randomly into NS, and formalin groups, and each group was divided into 1 h, 1 d, 3 d, 7 d and 14 d subgroups. The L5 segment of spinal cord was dissected out at the designed ending time point and sectioned in series in frozen preparation for iNOS and OX42 immuno- histochemistry.In all rats, the weighted pain score of the injected paw within 1 h after the formalin injection were measured according to the method described by Dubuisson and Dennis (1977). Moreover, thermal withdrawal latency and mechanical withdraw threshold of the injected area and the corresponding area in the paw contralateral to the injection were measured before the animals were sacrificed at the designed ending time points.Results: The rats showed characteristic flinch reflexes in the injected paw after the formalin injection. The flinches lasted 1 h or more. The thermal withdrawal latency and mechanical withdraw threshold were increased in the injected area, and were decreased in the corresponding area in the paw contralateral to the injection. A few of iNOS and GFAP double stained cells scattered in laminaeâ… -â…¡of the dorsal horn and the area around canalis centralis 1 h, 24 h, 48 h after the formalin injection. There was no significant difference in the numbers of the double-stained cells among 1 h, 24 h and 48 h groups. The comparison of iNOS and OX42 expression in series sections indicated that the iNOS immunoreactive cells were quite different from the OX42 immunoreactive cells in morphology, distribution and expression phase in the L5 segment of spinal cord after the formalin injection, and there was on significant coincidence between the expression of iNOS and OX42.Conclusion: Subcutaneous formalin injection produced a permanent hyperalgesia to heat and mechanical stimuli in the corresponding area in the paw contralateral to the injection, but an analgesia in the injected area.Glia might contribute in a certain degree, but not a major contributor, to the up-regulation of iNOS expression during formalin induced periphery inflammatory pain. The up- regulated iNOS expression during the process may be mediated mainly by neurons.