Differential involvement of trigeminal transition zone and laminated subnucleus caudalis in orofacial deep and cutaneous hyperalgesia: the effects of interleukin-10 and glial inhibitors.

BACKGROUND: In addition to caudal subnucleus caudalis (Vc) of the spinal trigeminal complex, recent studies indicate that the subnuclei interpolaris/caudalis (Vi/Vc) transition zone plays a unique role in processing deep orofacial nociceptive input. Studies also suggest that glia and inflammatory cytokines contribute to the development of persistent pain. By systematically comparing the effects of microinjection of the antiinflammatory cytokine interleukin (IL)-10 and two glial inhibitors, fluorocitrate and minocycline, we tested the hypothesis that there was a differential involvement of Vi/Vc and caudal Vc structures in deep and cutaneous orofacial pain. RESULTS: Deep or cutaneous inflammatory hyperalgesia, assessed with von Frey filaments, was induced in rats by injecting complete Freund's adjuvant (CFA) into the masseter muscle or skin overlying the masseter, respectively. A unilateral injection of CFA into the masseter or skin induced ipsilateral hyperalgesia that started at 30 min, peaked at 1 d and lasted for 1-2 weeks. Secondary hyperalgesia on the contralateral site also developed in masseter-, but not skin-inflamed rats. Focal microinjection of IL-10 (0.006-1 ng), fluorocitrate (1 microg), and minocycline (0.1-1 microg) into the ventral Vi/Vc significantly attenuated masseter hyperalgesia bilaterally but without an effect on hyperalgesia after cutaneous inflammation. Injection of the same doses of these agents into the caudal Vc attenuated ipsilateral hyperalgesia after masseter and skin inflammation, but had no effect on contralateral hyperalgesia after masseter inflammation. Injection of CFA into the masseter produced significant increases in N-methyl-D-aspartate (NMDA) receptor NR1 serine 896 phosphorylation and glial fibrillary acidic protein (GFAP) levels, a marker of reactive astrocytes, in Vi/Vc and caudal Vc. In contrast, cutaneous inflammation only produced similar increases in the Vc. CONCLUSION: These results support the hypothesis that the Vi/Vc transition zone is involved in deep orofacial injury and suggest that glial inhibition and interruption of the cytokine cascade after inflammation may provide pain relief.

Microinjection of IL-1ß into the trigeminal transition zone produces bilateral NMDA receptor-dependent orofacial hyperalgesia involving descending circuitry.

Our recent studies indicate that the prototypic proinflammatory cytokine IL-1ß is upregulated in astroglial cells in the trigeminal interplolaris/caudalis (Vi/Vc) transition zone, a region of the spinal trigeminal complex involved in trigeminal pain processing, after masseter muscle inflammation. Here we investigated the effect of microinjection of IL-1ß into the Vi/Vc transition zone on orofacial nociception. The mechanical sensitivity of the orofacial site was assessed with von Frey microfilaments. The EF(50) values, defined as the von Frey filament force (g) that produces a 50% response frequency, were derived and used as a measure of mechanical sensitivity. A significant reduction in EF(50) indicates the occurrence of mechanical hyperalgesia/allodynia. Unilateral intra-Vi/Vc IL-1ß (0.016-160 fmol) produced hyperalgesia/allodynia dose-dependently, which appeared at bilateral facial sites. The hyperalgesia was detectable as early as 30 min and lasted for 2-6 h (n=6, p<0.01). Intra-Vi/Vc pretreatment with an IL-1receptor antagonist (1 nmol) attenuated the IL-1ß-induced hyperalgesia (p<0.01). Pre-injection of AP-5 (10 pmol) and MK-801 (20 pmol), two NMDA receptor antagonists, significantly attenuated IL-1ß-induced hyperalgesia (p<0.05). Pretreatment with glial inhibitors fluorocitrate (120 pmol), minocycline (200 pmol) and propentofylline (10 pmol) did not attenuate IL-1ß-induced hyperalgesia. Excitotoxic lesions of the rostral ventromedial medulla with ibotenic acid (2 µg) abolished IL-1ß-induced contralateral hyperalgesia, suggesting a contribution of descending facilitatory drive. These results suggest that the IL-1ß-produced effect on nociception was downstream to glial activation and involves interaction with NMDA receptors.

Glial-cytokine-neuronal interactions underlying the mechanisms of persistent pain.

The emerging literature implicates a role for glia/cytokines in persistent pain. However, the mechanisms by which these non-neural elements contribute to CNS activity-dependent plasticity and pain are unclear. Using a trigeminal model of inflammatory hyperalgesia, here we provide evidence that demonstrates a mechanism by which glia interact with neurons, leading to activity-dependent plasticity and hyperalgesia. In response to masseter inflammation, there was an upregulation of glial fibrillary acidic proteins (GFAPs), a marker of astroglia, and interleukin-1beta (IL-1beta), a prototype proinflammatory cytokine, in the region of the trigeminal nucleus specifically related to the processing of deep orofacial input. The activated astroglia exhibited hypertrophy and an increased level of connexin 43, an astroglial gap junction protein. The upregulated IL-1beta was selectively localized to astrocytes but not to microglia and neurons. Local anesthesia of the masseter nerve prevented the increase in GFAP and IL-1beta after inflammation, and substance P, a prototype neurotransmitter of primary afferents, induced similar increases in GFAP and IL-1beta, which was blocked by a nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester. Injection of IL-1 receptor antagonist and fluorocitrate, a glial inhibitor, attenuated hyperalgesia and NMDA receptor phosphorylation after inflammation. In vitro application of IL-1beta induced NR1 phosphorylation, which was blocked by an IL-1 receptor antagonist, a PKC inhibitor (chelerythrine), an IP3 receptor inhibitor (2-aminoethoxydiphenylborate), and inhibitors of phospholipase C [1-[6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione] and phospholipase A2 (arachidonyltrifluoromethyl ketone). These findings provide evidence of astroglial activation by tissue injury, concomitant IL-1beta induction, and the coupling of NMDA receptor phosphorylation through IL-1 receptor signaling.

GABAA but not GABAB receptors in the rostral anterior cingulate cortex selectively modulate pain-induced escape/avoidance behavior.

The rostral anterior cingulate cortex (rACC) is involved in supraspinal nociceptive processing. ACC lesions relieve persistent pain, but do not affect the patient's ability to localize a noxious stimulus. Since the rACC has a high density of GABA receptors, it is possible that pain processing is influenced by these receptors in the rACC. The present experiments examined the involvement of rat rACC GABA(A) and GABA(B) receptors in regard to sensitivity to mechanical stimulation and escape/avoidance behavior in response to a noxious stimulus following L5 spinal nerve ligation. Rats were or were not afflicted with a neuropathic pain condition by an L5 spinal nerve ligation. rACC microinjection of 10 microg/microl GABA, a GABA(A) agonist (0.001 microg/microl, 0.1 microg/microl, or 0.5 microg/microl muscimol), a GABA(B) agonist (0.1 microg/microl, 1 microg/microl, or 5 microg/microl baclofen), or saline, did not alter mechanical withdrawal thresholds. Importantly, following 10 microg/microl GABA, 0.1 microg/microl, or 0.5 microg/microl muscimol microinjected into the rACC, place escape/avoidance behavior to a noxious mechanical stimulus was attenuated in injured animals. The attenuation was specific to the rACC and was blocked by a preadministered microinjection of the appropriate antagonist(s) into the rACC. In conclusion, microinjection of GABA and higher doses of muscimol did not decrease mechanical hyperalgesia but did attenuate place escape/avoidance behavior that is associated with mechanical stimulation of the ligated paw. These results provide additional support for the role of the rACC in higher order supraspinal processing of noxious events and suggest that rACC GABA(A) receptors significantly contribute to this processing.

Selective regulation of pain affect following activation of the opioid anterior cingulate cortex system.

Morphine and surgical cingulotomy, or transection of the anterior cingulate cortex (ACC), provides relief of chronic pain by selectively decreasing the affective dimension of the condition without altering sensory processing. Clinical reports suggest that morphine might be acting at the level of the ACC to alter the complex experience of pain. Therefore, the purpose of this experiment was to directly examine the functional role of the ACC in processing the aversive nature of pain induced by ligation of the L5 spinal nerve. Systemic administration of low dose morphine produced a selective attenuation of pain affect, as indicated by a decrease in the aversiveness of noxious cutaneous stimulation in nerve-damaged animals, with no alteration of mechanical paw withdrawal threshold. Supraspinally, microinjection of morphine into the ACC produced a selective naloxone reversible reduction in pain affect, as indicated by a decrease in the aversiveness of noxious cutaneous stimulation in nerve-damaged animals, with no alteration of response to mechanical stimulation. These data demonstrate the central role of the ACC opioid system in selectively processing the aversive quality of noxious mechanical stimulation in animals with a persistent pain condition.

Electrical stimulation of the primary somatosensory cortex inhibits spinal dorsal horn neuron activity.

Cortical stimulation has been demonstrated to alleviate certain pain conditions. The aim of this study was to determine the responses of the spinal cord dorsal horn neurons to stimulation of the primary somatosensory cortex (SSC). We hypothesized that direct stimulation of the SSC will inhibit the activity of spinal dorsal horn neurons by activating the descending inhibitory system. Thirty-four wide dynamic range spinal dorsal horn neurons were recorded in response to graded mechanical stimulation (brush, pressure, and pinch) at their respective receptive fields while a stepwise electrical stimulation (300 Hz, 0.1 ms, at 10, 20, and 30 V) was applied in the SSC through a bipolar tungsten electrode. The responses to brush at control, 10 V, 20 V, 30 V, and recovery were 16.0 +/- 2.3, 15.8 +/- 2.2, 14.6 +/- 1.8, 14.8 +/- 2.0, and 17.0 +/- 2.2 spikes/s, respectively. The responses to pressure at control, 10 V, 20 V, 30 V, and recovery were 44.7 +/- 5.5, 37.0 +/- 5.6, 29.5 +/- 4.8, 31.6 +/- 5.2, and 43.2 +/- 5.7 spikes/s, respectively. The responses to pinch at control, 10 V, 20 V, 30 V, and recovery were 58.1 +/- 7.0, 42.9 +/- 5.5, 34.8 +/- 3.9, 34.6 +/- 4.4, and 52.6 +/- 6.0 spikes/s, respectively. Significant decreases of the dorsal horn neuronal responses to pressure and pinch were observed during SSC stimulation. It is concluded that electrical stimulation of the SSC produces transient inhibition of the responses of spinal cord dorsal horn neurons to higher intensity mechanical stimuli without affecting innocuous stimuli.

Dorsal horn neuron response patterns to graded heat stimuli in the rat.

Sensory input from various receptors in the periphery first becomes integrated in the spinal cord dorsal horn. The response of the spinal cord dorsal horn neurons to mechanical stimuli are classified as low threshold, high threshold, and wide dynamic range neurons. However, the response pattern of deep dorsal horn cells to heat has not been well described. In this study, the response of the spinal cord dorsal horn neurons to graded heat stimuli were characterized in 147 neurons in rats by extracellular single cell recording. After a differentiable cell was identified, the Peltier heat stimulator was applied to the receptive field and the base temperature was set at 30 degrees C. The heat stimulus was delivered for 10 s from 37-51 degrees C in 2 degrees C increments, with an inter-stimulus interval of 30 s. Out of the 147 neurons, five statistically distinguishable response patterns were identified by latent class cluster analysis. It is concluded that variation of temperature may account for the observed results and indicate functionally different subsets of heat-responsive cells in the deep dorsal horn.

Electrical stimulation of the anterior cingulate cortex reduces responses of rat dorsal horn neurons to mechanical stimuli.

The anterior cingulate cortex (ACC) is involved in the affective and motivational aspect of pain perception. Behavioral studies show a decreased avoidance behavior to noxious stimuli without change in mechanical threshold after stimulation of the ACC. However, as part of the neural circuitry of behavioral reflexes, there is no evidence showing that ACC stimulation alters dorsal horn neuronal responses. We hypothesize that ACC stimulation has two phases: a short-term phase in which stimulation elicits antinociception and a long-term phase that follows stimulation to change the affective response to noxious input. To begin testing this hypothesis, the purpose of this study was to examine the response of spinal cord dorsal horn neurons during stimulation of the ACC. Fifty-eight wide dynamic range spinal cord dorsal horn neurons from adult Sprague-Dawley rats were recorded in response to graded mechanical stimuli (brush, pressure, and pinch) at their respective receptive fields, while simultaneous stepwise electrical stimulations (300 Hz, 0.1 ms, at 10, 20, and 30 V) were applied in the ACC. The responses to brush at control, 10, 20, and 30 V, and recovery were 14.2 +/- 1.4, 12.3 +/- 1.2, 10.9 +/- 1.2, 10.3 +/- 1.1, and 14.1 +/- 1.4 spikes/s, respectively. The responses to pressure at control, 10, 20, and 30 V, and recovery were 39.8 +/- 4.7, 25.6 +/- 3.0, 25.0 +/- 3.0, 21.6 +/- 2.4, and 34.2 +/- 3.7 spikes/s, respectively. The responses to pinch at control, 10, 20, and 30 V, and recovery were 40.7 +/- 3.8, 30.6 +/- 3.1, 27.8 +/- 2.8, 27.2 +/- 3.2, and 37.4 +/- 3.9 spikes/s, respectively. We conclude that electrical stimulation of the ACC induces significant inhibition of the responses of spinal cord dorsal horn neurons to noxious mechanical stimuli. The stimulation-induced inhibition begins to recover as soon as the stimulation is terminated. These results suggest differential short-term and long-term modulatory effects of the ACC stimulation on nociceptive circuits.

Behavioral evidence for competing motivational drives of nociception and hunger.

Homeostasis, an organisms' tendency to maintain a healthy balance of the physiological state of the body, is necessary for survival. Hunger induces a motivational state to consume food. Recently, pain has been referred to as a homeostatic emotion similar to hunger or thirst in that animals are motivated to respond in a certain way that may increase their chance of survival. Therefore, the purpose of the present experiment was to examine behavior in rodents during two competing homeostatic/motivational drives (i.e., hunger and formalin pain). During the first phase of the formalin test, animals displayed typical responsiveness to the inflammatory condition and completed fewer chains for food reinforcement as compared to the baseline session. However, during the second phase of the formalin test, animals showed decreased nociceptive behavior compared to formalin-injected animals that were not trained in the operant conditioning paradigm. During this phase, the trained animals exhibited maximal responsiveness for food reinforcement. These results demonstrate that the engagement of behaviors reflecting motivational drives to restore homeostasis depends on the intensity or degree of imbalance of the competing drives. More specifically, animals are motivated to attend to one state of imbalance at a time.

Effect of chronic vincristine treatment on mechanical withdrawal response and pre-pulse inhibition in the rat.

Chemotherapeutic agents are associated with a number of serious side-effects. In addition to the development of peripheral neuropathy, patients often complain of additional symptoms related to attentional mechanisms. Although a great deal of interest is directed towards understanding the mechanisms underlying the development of peripheral neuropathy, there is a paucity of research that has examined the extent of impairment of attention in animals receiving chemotherapeutic agents. Therefore, the purpose of this experiment was to examine attentional mechanisms using the method of pre-pulse inhibition in animals that were chronically treated with vincristine. Although vincristine treated animals developed signs of peripheral neuropathy, there was no associated alteration of pre-pulse inhibition relative to vehicle treated animals. These results highlight the importance of continuing to develop methodology to model symptom burden in patients receiving chemotherapy.

Differential effect of anterior cingulate cortex lesion on mechanical hypersensitivity and escape/avoidance behavior in an animal model of neuropathic pain.

Various limbic system structures have been implicated in processing noxious information. One such structure is the anterior cingulate cortex (ACC), a region that is thought to modulate higher order processing of noxious input related to the affective/motivational component of pain. The present experiment examined the involvement of the ACC in higher order pain processing by measuring paw withdrawal threshold and escape/avoidance responses in the L5 spinal nerve ligation model of neuropathic pain before and following electrolytic lesion of the ACC. In the place/escape avoidance paradigm, the afflicted paw is mechanically stimulated when the animal is in the preferred dark area of the chamber and the contralateral paw is stimulated when the animal is in the light area. Escape/avoidance was defined as a shift from the preferred dark area to an increase of time spent in the light area of the chamber. Animals with L5 ligation had significantly lower mechanical paw withdrawal threshold (hypersensitivity) and enhanced escape/avoidance behavior. ACC lesion in animals with L5 ligation did not alter mechanical hypersensitivity, but did significantly decrease escape/avoidance behavior. Anxiety, as measured using the light-enhanced startle paradigm, was not altered by ACC lesion. These results highlight the utility of novel behavioral test paradigms and provide additional support for the role of the ACC in higher order processing of noxious information, suggesting that ACC lesions selectively decrease negative affect associated with neuropathy-induced hypersensitivity.

Decreased L5 spinal nerve ligation nociceptive behavior following L4 spinal nerve transection.

This study used the escape/avoidance paradigm to explore the role of the L4 spinal nerve in L5 ligation nociception. Unlike L5-ligated controls, L5-ligated/L4-transected animals had normal mechanical withdrawal threshold and did not escape/avoid mechanical stimulation of the afflicted paw. The result from the escape/avoidance paradigm, which does not rely on a reflexive withdrawal response, directly supports the hypothesis that the L4 spinal nerve contributes to L5 ligation neuropathic pain.