Christopher W. Loomis

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

&NA; 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°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 &mgr;g), 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 &mgr;g) 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, &ggr;‐D‐glutamylglycine (DGG; 50 &mgr;g) 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.

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

&NA; 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 24 h, 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 PGE2. Thus, brushing the plantar surface of the affected hindpaw with a cotton‐tipped applicator, 5 days postligation, increased the [PGE2]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.

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

&NA; 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 GABAA‐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 &mgr;g in 0.1 &mgr;l) 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 ED50’s (95% CIs) 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 EP1‐ 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.

Comparable dose-dependent inhibition of AP-7 sensitive strychnine-induced allodynia and paw pinch-induced nociception by mexiletine in the rat.

Abstract The blockade of spinal glycine receptors with intrathecal (i.t.) strychnine produces segmentally‐localized allodynia in the rat; a reversible and highly reproducible effect that is attained without peripheral or central nerve injury. We investigated the effect of i.v. mexiletine, an orally active congener of lidocaine, on strychnine allodynia and compared the dose‐response relationship of mexiletine in normal (noxious paw pinch) versus abnormal (i.t. strychnine) nociceptive conditions. In addition, we determined the dose‐response effect of i.t. AP‐7 (an NMDA antagonist) on strychnine allodynia. Male, Sprague‐Dawley rats, fitted with chronic i.t. catheters, were lightly anesthetized with urethane. Stimulus evoked changes in blood pressure and heart rate were recorded from the left carotid artery and cortical electroencephalographic (EEG) activity was continuously monitored using subdermal needle electrodes. After i.t. strychnine (40 &mgr;g), repetitive brushing of the hair (hair deflection) evoked a progressive increase in mean arterial pressure and heart rate, an abrupt motor withdrawal response, and desynchronization of the EEG, equivalent to those elicited by the chemical nociceptive agent, mustard oil (without strychnine). Pretreatment with mexiletine (5–30 mg/kg i.v. 5 min before i.t. strychnine) dose‐dependently inhibited the responses evoked by noxious hind paw pinch (no strychnine) and hair deflection (after i.t. strychnine) with equal potency (ED50s=9.1–17 mg/kg). Below 30 mg/kg, this effect was achieved without a change in EEG synchrony (cortical activity reflecting the level of anesthesia) and without affecting motor efferent pathways. Strychnine allodynia was also significantly blocked by i.t. AP‐7. The ED50s and 95% confidence intervals were 1.1 &mgr;g (0.7–1.8) for mean arterial pressure, 1.7 &mgr;g (0.5–6.0) for heart rate, and 0.4 &mgr;g (0.07–2.0) for withdrawal duration. Cortical EEG synchrony was unchanged after i.t. AP‐7 consistent with a spinal site of action. The data indicate that: (i) robust allodynia can be selectively induced with i.t. strychnine in animals whose somatosensory systems are otherwise normal; (ii) sub‐anesthetic doses of i.v. mexiletine inhibit the abnormal responses to low‐threshold (A‐fiber) afferent input in the strychnine model of allodynia (i.e., in the absence of peripheral or central nerve injury) at doses which affect normal nociception; and (iii) in the presence of i.t. strychnine, low‐threshold afferent input activates a spinal NMDA‐receptor mediated process normally restricted to noxious afferent input. Systemic mexiletine may have an important spinal site of action in abnormal pain states.

Distal and deglutitive inhibition in the rat esophagus: Role of inhibitory neurotransmission in the nucleus tractus solitarii ☆ ☆☆

BACKGROUND & AIMS This study aimed to show the presence of deglutitive and distal inhibition in the rat esophagus and to differentiate the underlying neural mechanisms. METHODS Under urethane anesthesia, the pharyngoesophageal tract was fitted with water-filled balloons for luminal distention and pressure recording. Neural activity was recorded in the nucleus tractus solitarii subnucleus centralis and rostral nucleus ambiguous. RESULTS Distal esophageal distention evoked both rhythmic local contractions and burst discharges of ambiguous neurons that were simultaneously inhibited by a swallow or proximal esophageal distention. In subnucleus centralis interneurons, type I rhythmic burst discharges correlated with distal esophageal pressure waves and were suppressed by midthoracic esophageal distention; type II non-rhythmic excitatory responses, like type III inhibitory responses, were evoked by distention of either the thoracic or distal esophagus. When applied to the surface of the solitarius complex, bicuculline and, less effectively, strychnine suppressed distal inhibition, and 2-(OH)-saclofen and 3-aminopropylphosphonic acid were ineffective. None of the drugs tested, including systemic picrotoxin, affected deglutitive inhibition. CONCLUSIONS Distal and deglutitive inhibition are present in the rat esophagus. The former, unlike the latter, depends on activation of ligand-gated chloride channels associated with subnucleus centralis premotor neurons. Inhibitory aminoacidergic local interneurons are a probable source of type II responses.

Inhibition of strychnine-allodynia is mediated by spinal adenosine A1- but not A2-receptors in the rat

Intrathecal (i.t.) strychnine produces localized allodynia in the rat without peripheral or central nerve injury. Intrathecal CPA (A1-selective agonist) and CGS-21680 (A2-selective agonist) dose-dependently inhibited strychnine-allodynia but with a 50-fold difference in potency (0.02-0.07 vs. 2.7-3.1 microgram, respectively). The anti-allodynic effect of CPA and CGS was completely blocked by pretreatment with the A1-selective antagonist, DPCPX (10 microgram i.t. ), but unaffected by the A2-selective antagonist, CSC (2 microgram i.t. ). The results indicate that spinal A1-, but not A2-, receptors modulate abnormal somatosensory input in the strychnine model, and suggest a difference in spinal purinergic modulation in injury vs. non-injury models of allodynia.

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

&NA; 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 &mgr;g). 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.

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.

Effects of vagal cooling on esophageal cardiovascular reflex responses in the rat.

Distension of the distal esophagus in the anesthetized rat causes a vagally-mediated arterial pressor and tachycardia response. To investigate the nature of viscerosensory fibers in the afferent limb of this reflex, the present study was carried out in urethane-anesthetized rats that were subjected to graded cooling of both cervical vagal trunks in situ. Distal esophageal distension was applied for 20 s by means of a water-filled high compliance balloon. Vagal cooling to 9 degrees C abolished pressor responses and unmasked a depressor component during maximal distension. Cooling to 7.5 degrees C blocked this inhibitory component, well above the temperature known to block C-fibers. We conclude that the cardiovascular response to esophageal distension is triggered via at least two subpopulations of A(delta) type vagal afferents that project to brain stem nuclei regulating central vasomotor tone.

Intrathecal oxymetazoline does not produce neurotoxicity in the spinal cord of the rat.

To determine if intrathecal (i.t.) oxymetazoline (OXY) induces histological evidence of spinal neurotoxicity, male, Sprague-Dawley rats (300-450 g; implanted with an i.t. catheter) were treated with i.t. saline or 100 nmol OXY twice daily for 3 days, or 200 or 300 nmol OXY once daily for 3 days. Spantide (D-Arg1, D-Try7,9, Leu11-substance P; 0.067 nmol = 0.1 microgram, 0.167 nmol = 0.25 microgram or 0.334 nmol = 0.5 microgram) or capsaicin (0.164 mumol = 50 micrograms), given as a single i.t. injection, were used as positive controls. Animals were killed 12 h after the last injection of saline or OXY, and 72 h after spantide or capsaicin. Spinal cord sections (L1 and adjacent segments) were examined by light microscopy for changes in gross morphology, substance P-like immunoreactivity (SP-IR) and calcitonin gene related peptide-like immunoreactivity (CGRP-IR). All doses of i.t. OXY produced antinociception (tail-flick ED50 = 53.7 nmol, paw pressure withdrawal ED50 = 93.3 nmol). Rectal temperature decreased by 1.5-2.4 degrees C up to 12 h after 100 nmol of i.t. OXY. There were no signs of inflammation or necrosis, and no detectable loss or damage to either spinal afferents or motor neurons as judged by SP-IR and CGRP-IR structures in spinal cords of OXY-treated animals (all doses) as compared to i.t. saline controls. Spantide (0.1 microgram) had no antinociceptive or neurotoxic effect; 0.25 microgram induced irreversible loss of the TF reflex and transient hind limb paralysis; 0.5 microgram induced irreversible loss of TF and PP responses, permanent hind limb paralysis, bladder and bowel dysfunction. The spinal cords from these animals showed signs of extensive necrosis, cavitation, and haemorrhage in the ventral horn accompanied by a loss of CGRP-IR motor neurons. Capsaicin-treated rats exhibited a permanent loss of the TF but not the PP response and a marked reduction of SP-IR spinal afferents in the dorsal horn. It is concluded that i.t. OXY produces antinociception in the rat with no detectable spinal neurotoxicity as assessed by parameters which are sensitive to the neurotoxins, spantide and capsaicin.