Central sensitization of nociceptive neurons in rat medullary dorsal horn involves purinergic P2X7 receptors

Abstract

Central sensitization is a crucial process underlying the increased neuronal excitability of nociceptive pathways following peripheral tissue injury and inflammation. Our previous findings have suggested that extracellular adenosine 5'-triphosphate (ATP) molecules acting at purinergic receptors located on presynaptic terminals (e.g., P2X2/3, P2X3 subunits) and glial cells are involved in the glutamatergic-dependent central sensitization induced in medullary dorsal horn (MDH) nociceptive neurons by application to the tooth pulp of the inflammatory irritant mustard oil (MO). Since growing evidence indicates that activation of P2X7 receptors located on glia is involved in chronic inflammatory and neuropathic pain, the aim of the present study was to test in vivo for P2X7 receptor involvement in this acute inflammatory pain model. Experiments were carried out in anesthetized Sprague-Dawley male rats. Single unit recordings were made in MDH functionally identified nociceptive neurons for which mechanoreceptive field, mechanical activation threshold and responses to noxious stimuli were tested. We found that continuous intrathecal (i.t.) superfusion over MDH of the potent P2X7 receptor antagonists brilliant blue G and periodated oxidized ATP could each significantly attenuate the MO-induced MDH central sensitization. MDH central sensitization could also be produced by i.t. superfusion of ATP and even more effectively by the P2X7 receptor agonist benzoylbenzoyl ATP. Superfusion of the microglial blocker minocycline abolished the MO-induced MDH central sensitization, consistent with reports that dorsal horn P2X7 receptors are mostly expressed on microglia. In control experiments, superfusion over MDH of vehicle did not produce any significant changes. These novel findings suggest that activation of P2X7 receptors in vivo may be involved in the development of central sensitization in an acute inflammatory pain model.

Figure 1

Diagram of experimental paradigms.

Figure 2

Effects of continuous i.t. superfusion of oATP, BBG and PBS on the neuroplastic changes in MDH induced by MO application to the tooth pulp. A. Changes in NS neuronal RF size. Note that during PBS superfusion, MO application produced significant increases in pinch RF size throughout the 50-min observation period (P<0.001, RM ANOVA; *P<0.05, Dunnett's test; n=6), and that superfusion of oATP and BBG only partially but still significantly attenuated the MO-induced increases in neuronal RF size (P<0.001, RM ANOVA, both n=6). B. Changes in neuronal responses to graded mechanical stimuli. C. Changes in mechanical activation threshold of NS neurons. Note that during PBS superfusion, MO application produced significant increases in responses and decreases in threshold (P<0.001 and P<0.05, respectively, RM ANOVA; *P<0.05, Dunnett's test), and that superfusion of oATP and BBG significantly blocked the MO-induced increases in responses and decrease in threshold (both P>0.5, RM ANOVA). All these MO-induced neuroplastic changes in the oATP and BBG groups were significantly less than those in the PBS group (all P<0.001, 2-way ANOVA). Post-hoc analysis indicated that there were significant differences in values at most post-MO time-points between the PBS and these two groups (#P<0.05, Dunnett test). D. The recording sites of the NS neurons in PBS, oATP and BBG groups were histologically retrieved in the deep laminae of MDH.

Figure 3

Examples showing MO-induced neuroplastic changes (central sensitization) in NS neurons after continuous i.t. superfusion of PBS, BBG and oATP over MDH. For each example, the pinch RF (upper panel), activation threshold (middle panel) and responses to pinch/pressure stimuli (lower panel) are shown. Data at 2 h after PBS or BBG or oATP superfusion (baseline, i.e., prior to MO application), 18 min and 48 min after MO application of each NS neuron are arranged in columns from left to right. Note that the NS neurons illustrated from the PBS group showed MO-induced neuroplastic changes, including an appearance of a novel tactile RF at 18 min after MO application, while no marked changes occurred following MO application in the NS neuron of the BBG and oATP group. The neuronal discharges are displayed in an interspike instantaneous frequency distribution. The activation threshold of a given neuron is marked by a short horizontal bar at the intersection of the cut-off (vertical) line and the ascending force curve. The unit responses to pinch/pressure stimuli (1-2 times threshold intensity) are also shown in the instantaneous frequency distribution during the 5 s stimulation period. Black area indicates the pinch/pressure RF. T & P RF refers to that part of the RF from which the neuron could be activated by tactile as well as pinch/pressure stimulation.

Figure 4

A. Dose-dependent responses of i.t. superfusion of BzATP that compare with the MO-induced central sensitization in MDH. After the pretreatment of a mixture of suramin and DPCPX for 25 min, BzATP (30, 100, or 300 μM) was superfused on the medulla for 15 min. BzATP produced dose-dependently central sensitization-like responses (slow onset and gradual buildup), reflected as increases in pinch RF size expansion (A1), decrease in activation threshold (A2), and increases in responses to mechanical stimuli (A3). B. Comparison between superfusion of BzATP and ATP (at same doses) of chemical-evoked changes in RF size, activation threshold and pinch responses in MDH. Note that at the same 100 μM concentration, ATP evoked novel tactile RF in NS neurons which have been defined as lack of tactile RF in normal conditions, while BzATP did not evoke it (#P<0.05, Fisher test, n=5; B1) and was more effective also in reducing activation threshold (B3); in contrast, BzATP produced significantly stronger expansion in pinch RF size than that produced by ATP (#P<0.05, t-test, n=5; B2). Regarding pinch-evoked responses, BzATP also was much more potent than ATP (#P<0.05, t-test, n=5; B 4). For all figures in A and B, *P<0.05 represents significant difference between the value of post-BzATP or post-ATP time-points and the baseline values (1-way RM ANOVA or ANOVA on Ranks, n=5-6).

Figure 5

Effects of continuous i.t. superfusion of minocycline and PBS on the neuroplastic changes in MDH induced by MO application to the tooth pulp. The MO-induced changes in NS neuronal RF size (A), responses to graded mechanical stimuli (B) and mechanical activation threshold (C). Note that during PBS superfusion, MO application produced significant increases in pinch RF size, responses to graded stimuli, and decreases in threshold (P<0.05-0.001, RM ANOVA; *P<0.05, Dunnett's test; n=6) throughout the 50-min observation period, and that minocycline superfusion significantly blocked the MO-induced changes in neuronal properties (P>0.1-0.3, RM ANOVA or ANOVA on Ranks, n=6). All these MO-induced neuroplastic changes in the minocycline group were significantly less than those in the PBS group (all P<0.001, 2-way ANOVA). Post-hoc analysis indicated that there were significant differences in values at most post-MO time-points between these two groups (#P<0.05, Dunnett test). D. The recording sites of the NS neurons in PBS and minocycline groups were histologically retrieved in the deep laminae of MDH.