Spinal prostaglandins facilitate exaggerated A- and C-fiber-mediated reflex responses and are critical to the development of allodynia early after L5-L6 spinal nerve ligation.

BACKGROUND: Spinal prostaglandins are important in the early pathogenesis of spinal nerve ligation (SNL)-induced allodynia. This study examined the effect of SNL on the expression of cyclooxygenase (COX)-1, COX-2, and prostaglandin E2 receptors in the rat lumbar spinal cord, and the temporal and pharmacologic relation of these changes to the exaggerated A- and C-fiber-mediated reflex responses and allodynia, 24 h after injury. METHODS: Male Sprague-Dawley rats, fitted with intrathecal catheters, underwent SNL or sham surgery. Paw withdrawal threshold, electromyographic analysis of the biceps femoris flexor reflex, and immunoblotting of the spinal cord were used. RESULTS: Both allodynia (paw withdrawal threshold of < or = 4 g) and exaggerated A- and C-fiber-mediated reflex responses (i.e., decrease in activation threshold, increase in evoked activity, including windup; P < 0.05) were evident 24 h after SNL but not sham surgery. Allodynic animals exhibited significant increases in prostaglandin E2 receptor (subtypes 1-3) and COX-1 (but not COX-2) expression in the ipsilateral lumbar dorsal horn. The corresponding ventral horns and contralateral dorsal horn were unchanged from sham controls. Exaggerated A- and C-fiber-mediated reflex responses were significantly attenuated by intrathecal SC-560 or SC-51322, but not SC-236, given 24 h after SNL. CONCLUSION: These results provide further evidence that spinal prostaglandins, derived primarily from COX-1, are critical in the exaggeration of A- and C-fiber input and allodynia, 24 h after SNL.

Increased expression of cyclooxygenase and nitric oxide isoforms, and exaggerated sensitivity to prostaglandin E2, in the rat lumbar spinal cord 3 days after L5-L6 spinal nerve ligation.

BACKGROUND: Spinal prostaglandins seem to be important in the early pathogenesis of experimental neuropathic pain. Here, the authors investigated changes in the expression of cyclooxygenase and nitric oxide synthase (NOS) isoforms in the lumbar, thoracic, and cervical spinal cord and the pharmacologic sensitivity to spinal prostaglandin E2 (PGE2) after L5-L6 spinal nerve ligation (SNL). METHODS: Male Sprague-Dawley rats, fitted with intrathecal catheters, underwent SNL or sham surgery 3 days before experimentation. Paw withdrawal threshold was monitored for up to 20 days. Immunoblotting, spinal glutamate release, and behavioral testing were examined 3 days after SNL. RESULTS: Allodynia (paw withdrawal threshold < or = 4 g) was evident 1 day after SNL and remained stable for 20 days. Paw withdrawal threshold was unchanged (P > 0.05) from baseline (> 15 g) after sham surgery except for a small but significant decrease on day 20. Cyclooxygenase 2, neuronal NOS, and inducible NOS were significantly increased in the ipsilateral lumbar dorsal horn after SNL. Expression in the contralateral dorsal horn and ventral horns (lumbar segments) or bilaterally (thoracic and cervical segments) was unchanged from sham controls. This was accompanied by a significant decrease in both the EC50 of PGE2-evoked glutamate release and the ED50 of PGE2 on brush-evoked allodynia. Enhanced sensitivity to PGE2 was localized to lumbar segments of SNL animals and attenuated by SC-51322 or S(+)-ibuprofen, but not R(-)-ibuprofen (100 mum). CONCLUSION: The increased expression of cyclooxygense-2, neuronal NOS, and inducible NOS and the enhanced sensitivity to PGE2 in spinal segments affected by SNL support the hypothesis that spinal prostanoids play an early pathogenic role in experimental neuropathic pain.

Inhibition of spinal prostaglandin synthesis early after L5/L6 nerve ligation prevents the development of prostaglandin-dependent and prostaglandin-independent allodynia in the rat.

BACKGROUND: Prostaglandins, synthesized in the spinal cord in response to noxious stimuli, are known to facilitate nociceptive transmission, raising questions about their role in neuropathic pain. The current study tested the hypothesis that spinal nerve ligation-induced allodynia is composed of an early prostaglandin-dependent phase, the disruption of which prevents allodynia. METHODS: Male Sprague-Dawley rats, fitted with intrathecal drug delivery or microdialysis catheters, underwent left L5-L6 spinal nerve ligation or sham surgery. Paw withdrawal threshold, brush-evoked behavior, and the concentration of prostaglandin E2 (PGE2) in spinal cerebrospinal fluid ([PGE2]dialysate) were determined for up to 24 days. PGE2-evoked glutamate release from spinal slices was also determined. RESULTS: Paw withdrawal threshold decreased from at least 15 g (control) to less than 4 g, beginning 1 day after ligation. Brushing the affected hind paw evoked nociceptive-like behavior and increased [PGE2]dialysate (up to 257 +/- 62% of baseline). There was no detectable change in basal [PGE2]dialysate from preligation values. The EC50 of PGE2-evoked glutamate release (2.4 x 10-11 M, control) was significantly decreased in affected spinal segments of allodynic rats (8.9 x 10-15 M). Treatment with intrathecal S(+)-ibuprofen or SC-560, beginning 2 h after ligation, prevented the decrease in paw withdrawal threshold, the brush-evoked increase in [PGE2]dialysate, and the change in EC50 of PGE2-evoked glutamate release. R(-)-ibuprofen or SC-236 had no effect. CONCLUSIONS: The results of this study provide solid evidence that spinal prostaglandins, synthesized by cyclooxygenase-1 in the first 4-8 h after ligation, are critical in the pathogenesis of prostaglandin-dependent and prostaglandin-independent allodynia and that their early pharmacologic disruption affords protection against this neuropathic state in the rat.

Spinal nerve injury activates prostaglandin synthesis in the spinal cord that contributes to early maintenance of tactile allodynia.

To determine if spinal prostaglandins (PG) contribute to tactile allodynia, male, Sprague-Dawley rats were fitted with either intrathecal (i.t.) microdialysis or drug delivery catheters 3 days before tight ligation of the left lumber 5/6 spinal nerves. Paw withdrawal thresholds (PWT) were determined using von Frey filaments. Ligated rats developed tactile allodynia within 24h, as evidenced by a decrease in PWT in the affected hindpaw (<4 g vs. >15 g control). Sham-operated controls were unchanged from baseline (>15 g). Allodynia was also characterized by a significant increase in the evoked release of PGE(2). Thus, brushing the plantar surface of the affected hindpaw with a cotton-tipped applicator, 5 days postligation, increased the [PGE(2)](dialysate) to 199+/-34% of the prestimulus control period. In contrast, brushing had no detectable effect on release before surgery or in sham-operated animals. Basal release (no brushing) was similar before and after surgery (sham-operated and ligated rats). In a separate group of rats and beginning 2 days after ligation, the acute i.t. injection of S(+)-ibuprofen, SC-51322, SC-236, or SC-560 significantly reversed allodynia (maximum effect=69+/-9, 66+/-6, 57+/-4, 20+/-5%, respectively). R(-)-ibuprofen or vehicle were without effect. The results of this study suggest that: (a). spinal PG synthesis and allodynia-like behaviour are triggered by normally innocuous brushing after spinal nerve ligation; (b). pharmacological disruption of this cascade significantly reverses allodynia; (c). COX-2 is the relevant isozyme; and (d). the PG effect is mediated by spinal EP receptors.

Mediation by nucleus tractus solitarii glutamatergic neurotransmission of the cardiovascular reflex evoked by distal esophageal distension.

Distention of the rat distal esophagus evokes an arterial pressor and a cardioaccelerator response that depends upon activation of a vagal afferent projection to the nucleus tractus solitarii (NTS). The present study aimed to determine in urethane-anesthetized rats if the afferent limb of this reflex (a) relays in the NTS subdivision known ro receive esophageal afferents, and (b) utilizes glutamatergic synapses. To this end, tetrodotoxin or the glutamate antagonists gamma-D-glutamyl-glycine, 6-7-dinitroquinoxaline-2,3-dione (DNQX) and 2-amino-5-phosphonovaleric acid (AP-5) were applied to the NTS extraventricular surface rostral to the obex. All four agents inhibited both components of the reflex. DNQX or AP-5 produced a similar reversible inhibition upon pressure ejection in the vagal esophageal afferent projection area. Application of tetrodotoxin to the dorsomedial medulla oblongata caudal to the area postrema (AP) was ineffective. Basal heart rate (HR) (except in the case of AP-5) and blood pressure increased upon NTS surface application of the antagonists but not after intra-NTS ejection. At corresponding dorsal NTS sites, focal excitation of solitarial neurons with glutamate evoked hypotension and cardiac slowing. At adjacent ventral sites in the NTS subnucleus centralis (NTSc) and/or its immediate vicinity, glutamate elicited an arterial pressor response that coincided with an esophageal contraction in most but not all cases. In conclusion, afferent fibers of the esophago-cardiovascular reflex (ECVR) probably (1) terminate in the vicinity of esophageal premotor neurons comprising the NTSc and (2) activate second-order neurons via glutamate receptors of both the NMDA and non-NMDA subtype.

Topical bicuculline to the rat spinal cord induces highly localized allodynia that is mediated by spinal prostaglandins.

The purpose of this study was to investigate the allodynic effect of bicuculline (BIC) given topically to the dorsal surface of the rat spinal cord, and to determine if spinal prostaglandins (PGs) mediate the allodynic state arising from spinal GABA(A)-receptor blockade. Male Sprague-Dawley rats (325-400 g) were anaesthetized with halothane and maintained with urethane for the continuous monitoring of blood pressure (MAP), heart rate (HR) and cortical electroencephalogram (EEG). A laminectomy was performed to expose the dorsal surface of the spinal cord. Unilateral application of BIC (0.1 microg in 0.1 microl) to the L5 or L6 spinal segment induced a highly localized allodynia (e.g. one or two digits) on the ipsilateral hind paw. Thus, hair deflection (brushing the hair with a cotton-tipped applicator) in the presence, but not absence of BIC, evoked an increase in MAP and HR, abrupt motor responses (MR; e.g. withdrawal of the hind leg, kicking, and/or scratching) on the affected side, and desynchrony of the EEG. BIC-allodynia was dose-dependent, yielding ED(50)'s (95% CI's) of 45 ng (31-65) for MAP; 68 ng (46-101) for HR and 76 ng (60-97) for MR. Allodynia was sustained for up to 2 h with repeated BIC application without any detectable change in the location or area of peripheral sensitization. Pretreatment with either the EP(1)- receptor antagonist, SC-51322, the cyclooxygenase (COX)-2 selective inhibitor, NS-398, or the NMDA-receptor antagonist, AP-7, inhibited BIC-allodynia in a dose-dependent manner. The results demonstrate: (a) BIC, applied to the dorsal surface of the spinal cord, induces highly localized allodynia; (b) this effect can be sustained with repeated BIC application; (c) it is evoked by NMDA-dependent afferent input; (d) spinal PGs are synthesized by constitutive COX-2 during BIC-allodynia; and (e) spinal PGs contribute to the abnormal processing of tactile input via spinal EP1-receptors.

Milacemide, a glycine pro-drug, inhibits strychnine-allodynia without affecting normal nociception in the rat.

The blockade of spinal glycine receptors with intrathecal (i.t.) strychnine (STR) produces reversible, segmentally localized allodynia in the rat. The purpose of this study was: (1) to investigate the effect of the anticonvulsant agent, milacemide, a glycine pro-drug on STR-allodynia; (2) to compare this effect with that of milacemide on normal nociception (without STR); and (3) to determine the sensitivity of the anti-allodynic effect of milacemide to pretreatment with selective monoamine oxidase (MAO)-A (clorgyline) and MAO-B (L-deprenyl) inhibitors. Male Sprague-Dawley rats, fitted with chronic i.t. catheters, were lightly anesthetized with urethane. Hair deflection (HD) evoked maximum changes in blood pressure and heart rate were recorded from left carotid artery, and cortical electroencephalographic (EEG) activity was continuously monitored using subdermal needle electrodes before and after i.t. STR (40 microg). Rats were pretreated with a single intravenous (i.v.) injection of milacemide (100-600 mg/kg), 1 h before i.t. STR. To sustain the allodynic state, STR was injected every hour for up to 4 h. HD was applied to the affected dermatomes (2 min duration) using a cotton-tipped applicator at 5-min intervals for the duration of the STR effect. Normally innocuous HD elicited a marked increase in mean arterial blood pressure and heart rate, an immediate motor responses, and desynchronisation of EEG when applied to the cutaneous dermatomes affected by i.t. STR. Milacemide (100-600 mg/kg, i.v.) dose-dependently inhibited the heart rate and pressor responses (ED50 = 398 mg/kg; 95%CI = 196-873) and the motor responses (ED50 = 404 mg/kg; 95%CI = 275-727). Maximum inhibition was observed approximately 2 h after i.v. injection. The duration of action ranged from 3 h (400 mg/kg) to 4 h (600 mg/kg). Milacemide had no effect on the percent synchrony in the EEG. At the time of maximum inhibition of STR-allodynia (2 h post-infusion), responses evoked by noxious pinch were unaffected by milacemide. Pretreatment with L-deprenyl (3 mg/kg, i.p.), but not clorgyline (10 mg/kg, i.p.) significantly blocked the anti-allodynic effect of milacemide (600 mg/kg i.v). These data indicate that i.v. milacemide significantly attenuates the allodynia arising from spinal glycine receptor blockade, and are consistent with: (1) the selective modulation of low threshold afferent input by STR-sensitive, glycine interneurons in the rat spinal cord; and (2) the pharmacological actions of milacemide as a glycine pro-drug.

Morphine insensitive allodynia is produced by intrathecal strychnine in the lightly anesthetized rat.

The acute blockade of spinal glycinergic inhibition with intrathecal strychnine (i.t. STR; a glycine antagonist) in rats induces a change in somatosensory processing which is very similar to the sensory dysesthesia of clinical neural injury pain. In the present study, the effects of i.t. STR were examined in urethane-anesthetized rats. Noxious paw pinch (PP) or tail immersion (TI) in 55 degree C water evoked a pronounced pressor response, increased heart rate (HR) and desynchronized the electroencephalogram; a non-noxious, hair deflection (HD) elicited only minor cardiovascular responses. After i.t. STR (40 micrograms), an identical HD stimulus evoked markedly enhanced cardiovascular responses, resembling those evoked by noxious stimuli, and a HD-evoked motor withdrawal was observed. Consistent STR-dependent responses were only observed if a light plane of anesthesia was maintained for the duration of the experiment. The effects of i.t. STR were dose-dependent and reversible, lasting 15-30 min. Spinal morphine (50 micrograms) completely abolished the cardiovascular responses to PP and TI, but the HD-evoked, STR-dependent cardiovascular and motor withdrawal responses remained unchanged. In contrast, the non-selective excitatory amino acid antagonist, gamma-D-glutamylglycine (DGG; 50 micrograms) was effective in suppressing both the STR-dependent cardiovascular and motor withdrawal responses. These data suggest that STR-dependent responses evoked by non-noxious stimuli are mediated by mechanisms distinct from those of conventional noxious stimuli and that i.t. STR may be useful for investigating the spinal pharmacology of somatosensory processing following the loss of spinal glycinergic inhibition.