Peripheral And Spinal NMDA Receptors Are Involved In Spinal Microglia Activation And Long-term Hyperalgesia Induced By Formalin Injection

The formalin test has been widely used as an acute inflammatory model, characterized by biphasic behavioral responses (lasting for 1 to 2 hours) named acute and tonic phase, which could be quantitively measured by accounting the time for lifting and licking towards the injected place or by other behavioral responses. Using this model, previous studies from our lab and other investigators have indicated that NMDA receptors not only contribute to the initiation of afferent noxious signals originating from the peripheral nociceptive terminals but also the synaptic transmission of noxious inputs at the spinal level. However, recent evidence shows that subcutaneous injection of formalin could also produce dramatically a long-term hyperalgesia (LTH) even lasting for several days, in addition to a strong activation of spinal microglia lasting the similar period. The aim of the present study was designed to investigate if peripheral and spinal NMDA receptors were involved in the formalin-induced LTH by observing the effects of peripheral or intrathecal application of MK-801 on behavioral responses examined by thermal paw withdrawal latency (PWL), and the alterations in activation of microglia in the spinal cord during this period by immunocytochemical examination. There were two parts of experiments. The first part of experiments were designed for examining if peripheral NMDA receptors were involved in functioning of peripheral terminals to producing afferent nociceptive inputs that in turn affected spinal LTH and the activation of spinal microglia. In first set of experiments, animals were divided in 3 groups (n=6 in each) and all animals received formalin injections into the plantar aspects of two hind paws while three dosages of MK-801 (10 nmol, 30 nmol, and 50 nmol) were injected into the left hind paw 15 min before formalin injection with the same volumes of NS being injected into the right side in these groups, and the drug or NS injections were carried out repetitively in 4 consecutive days as before in these groups, respectively; then all rats were killed 7 days later for immunocytochemical examination of OX-42, a specific marker of microglia activation, in the spinal dorsal horn. The second set of experiments were similar to that of first one, but with three larger dosages of MK-801 (50 nmol, 100 nmol, and 150 nmol) or NS being injected only once at the first day into left or right side, respectively, 15 min before formalin injections in 3 groups (n=6); and then the immunocytochemistry was carried out 7 days later. The animals in the third set of experiments of this part were divided into two groups (n=6 in each): the experimental (MK-801+formalin) group, in which MK-801 (30 nmol) being injected into the dorsal aspect of the left hind paw 15 min before the formalin injection into the same site, and the same dose of MK-801 was injected once daily in the following 4 days; and the control (NS+formalin) group with 5 days'NS injections with formalin injection once at the first day; at the same time, PWL in all rats of two groups were examined first to get the baseline value before drug injection at the first day and in following 10 days (tests in former 4 days were carried out just before that day's drug injection). The second part of experiments were designed to observe the possible involvement of spinal NMDA receptors in the activation of microglia in the spinal dosal horn. Animals were divided into two groups (n=6 in each): intrathecal (i.t.) injection of MK-801 (10 nmol) or NS were carried out in rats of experimental group or control group once a day in 4 consecutive days, while formalin was injected into the plantar aspect of the left hindpaw of all rats 15 min after the pretreatment of MK-801 or NS at the first day, and then animals were killed 7 days later after formalin injection for immunocytochemical detection of OX-42 staining in the spinal dorsal horn. The results of experiments stated above showed that: 1) peripheral injection of formalin induced LTH in control group without either peripheral orintrathe...