Vjekoslav Miletic

Iontophoretic application of calcitonin gene-related peptide produces a slow and prolonged excitation of neurons in the cat lumbar dorsal horn

Calcitonin gene-related peptide (CGRP) was applied by iontophoresis onto physiologically characterized neurons. CGRP (20-100 nA) activated both wide-dynamic-range (5/8) and low-threshold mechanoreceptive units (3/12), but had no effect on nociceptive-specific neurons (0/4). The excitation was of slow onset (30 s to 3 min) and prolonged duration (up to 10 min). In none of the tested units did CGRP cause inhibition. The slow and prolonged action suggests a neuromodulatory role for CGRP in spinal cord sensory processes.

Increases in the concentration of brain derived neurotrophic factor in the lumbar spinal dorsal horn are associated with pain behavior following chronic constriction injury in rats

Animals exhibiting thermal hyperalgesia as a sign of neuropathic pain 7 days after loose ligation of the sciatic nerve exhibited a significant increase in the concentration of brain derived neurotrophic factor (BDNF) in their lumbar spinal dorsal horn. In contrast, following the disappearance of thermal hyperalgesia 28 days after loose ligation of the sciatic nerve, there were no differences in BDNF levels between control animals and those with sciatic ligations. These data suggest a close association in the timeline of the development and disappearance of behavioral signs of neuropathic pain with changes in BDNF levels in the lumbar spinal dorsal horn, and lend further support to the notion that plasticity in the processing of sensory information in the spinal dorsal horn may contribute to the development of persistent pain.

Loose ligation of the sciatic nerve is associated with TrkB receptor-dependent decreases in KCC2 protein levels in the ipsilateral spinal dorsal horn.

&NA; Significant decreases in the protein levels of potassium‐chloride co‐transporter 2 (KCC2) were detected in the ipsilateral spinal dorsal horn 4 h following loose ligation of the sciatic nerve. These decreases were associated with a change in hindlimb weight distribution suggestive of pain behavior. In contrast, no changes in GABA‐A receptor subunit alpha‐1 levels were detected. The decreases in KCC2 coincided with a significant ipsilateral increase in BDNF protein levels. Both the decreases in KCC2 levels and the early pain behavior were prevented by intrathecal pre‐treatment with the BDNF‐sequestering TrkB/Fc chimera protein or the tyrosine kinase blocker K252a. The ligation‐associated decreases in KCC2 levels were transient. In the ipsilateral spinal dorsal horn of ligated animals exhibiting weight‐bearing pain behavior 7 days after the ligation the KCC2 levels were identical to those in control or sham‐operated animals. These data suggested that TrkB‐dependent reduction in KCC2 protein levels in the spinal dorsal horn was an early consequence of peripheral nerve injury. This decrease in KCC2 may have elicited an early increase in overall dorsal horn neuronal excitability perhaps through a loss of GABA inhibition which is critically dependent on KCC2 activity. The increased neuronal excitability may in turn have caused enhanced and exaggerated communication between primary afferents and dorsal horn neurons to contribute to the early behavioral signs of pain.

Increases in the phosphorylation of cyclic AMP response element binding protein (CREB) and decreases in the content of calcineurin accompany thermal hyperalgesia following chronic constriction injury in rats.

&NA; Plasticity in the spinal dorsal horn may underlie the development of chronic pain following peripheral nerve injury or inflammation. In this study, we examined whether chronic constriction injury of the sciatic nerve was associated with changes in the immunoreactive content of cyclic AMP response element binding protein (CREB), protein kinase A (PKA), and calcineurin A&agr; and A&bgr; in the spinal dorsal horn. In animals exhibiting thermal hyperalgesia as a behavioral sign of neuropathic pain 7 days after loose ligation of the sciatic nerve (chronic constriction injury), there was a significant increase in the content of phosphorylated (activated) CREB (pCREB). In contrast, following the typical disappearance of thermal hyperalgesia 28 days after loose ligation surgery, there were no differences in pCREB content between control and sciatic ligation animals. The increased CREB activation associated with thermal hyperalgesia was accompanied by significant decreases in the content of both calcineurin A&agr; and A&bgr;. In contrast, there were no differences in the content of non‐phosphorylated CREB, and phosphorylated or non‐phosphorylated PKA between control and sciatic ligation animals either 7 or 28 days after surgery. These data established a close association in the expression of thermal hyperalgesia with CREB activation and decreased calcineurin content in the spinal dorsal horn. The data revealed a significant but reversible shift in the manner in which spinal neurons processed sensory information following peripheral nerve injury, and lent further support to the notion that plasticity in the spinal dorsal horn may have contributed to the development of chronic pain.

Responses of neurons in the rat nucleus submedius to noxious and innocuous mechanical cutaneous stimulation.

Extracellular recordings were used to characterize responses to cutaneous mechanical stimulation of 78 neurons in the rat nucleus submedius (SM). Thirty-nine of these units were activated by some type of cutaneous mechanical stimulation. Eighteen cells were activated exclusively by noxious stimuli. In 13 of these cells, responses were of swift onset and relatively rapid termination following stimulus application. In contrast, in three neurons responses were delayed both in onset and termination, and in two the response was immediate, but the markedly increased evoked activity outlasted stimulus application by 13 min. Receptive fields (RFs) of these nociceptive neurons were generally large, although none were bilateral. Four SM neurons were activated by innocuous stimuli, but their maximal response was obtained only after noxious stimulation. Responses of all of these neurons were of immediate onset and recovery, and their RFs were large (two were bilateral). Twelve SM neurons were activated maximally by innocuous stimuli. Responses of seven of these cells were immediate in onset and termination, while that of three were delayed in both onset and termination. Two of the 12 innocuous-only neurons quickly became unresponsive to repeated stimulus applications, and could be reactivated only after a rest period during which no stimuli were applied. RFs of these units were also generally large, and in three cases were bilateral. Five SM neurons responded by decreasing, or completely ceasing, their firing subsequent to noxious-only (n = 2), or innocuous-only (n = 3) stimulation. Four of these units had large RFs (two were bilateral). The remaining 39 SM neurons could not be activated by any type of mechanical cutaneous stimulation we tried. Electrical stimulation of the ventrolateral orbital cortex (VLO) was employed to examine frontal cortical projections of 21 SM neurons. Ten of these units were activated, although all of them synaptically rather than antidromically, and two were inhibited. There was no clear-cut relationship between neuronal location, physiological type, RF site, or VLO stimulation effects among the 39 SM neurons. These results provide further support for the involvement of SM neurons in nociceptive information signaling, and suggest additionally that the role of the nucleus is not limited to nociception but encompasses a wider range of cutaneous sensations.

The effect of continuous morphine analgesia on chronic thermal hyperalgesia due to sciatic constriction injury in rats

We employed hindfoot withdrawal latencies to radiant heat to assess the analgesic effect of prolonged morphine infusion on thermal hyperalgesia induced by chronic constriction injury (CCI) of the rat sciatic nerve. All CCI rats developed thermal hyperalgesia while sham-operated animals did not. Continuous systemic infusion of morphine dose-dependently reversed the thermal hyperalgesia in the CCI rats. In contrast, thermal hyperalgesia persisted in saline-treated CCI rats. Tolerance to morphines analgesic effect did not develop over a period of seven days of morphine infusion, which is considered long-term for animal models. These data suggest that morphine acts rapidly and effectively to reduce behavioral signs of hyperalgesia in rats with sciatic CCI, without the concomitant development of tolerance. Scheduled administration of morphine might be an appropriate treatment regimen for relief of neuropathic pain, and the infrequent use of opioids in equivalent human clinical pain syndromes due to fear of opioid unresponsiveness and tolerance might need to be re-evaluated.

Continual systemic infusion of lidocaine provides analgesia in an animal model of neuropathic pain.

&NA; We examined whether continual constant‐rate infusion of lidocaine would provide analgesia during the initial post‐injury phase in the chronic constriction injury model of neuropathic pain. Male Sprague–Dawley rats were divided into control and ligated groups and infused with saline or lidocaine (0.15, 0.33, 0.67, and 1.3 mg/kg/h) via subcutaneously implanted Alzet® osmotic minipumps. Thermal withdrawal latencies were obtained prior (Day 0) and 3 days after loose sciatic ligation and pump implantation surgery. Ligated animals receiving lidocaine at 0.67 or 1.3 mg/kg/h exhibited no change in withdrawal latency on Day 3 after surgery, indicating that lidocaine at these doses prevented the development of thermal hyperalgesia as a sign of neuropathic pain. In contrast, ligated animals treated with saline or lidocaine at 0.15 or 0.33 mg/kg/h exhibited hyperalgesia on Day 3 after surgery, indicating that these lower doses of lidocaine failed to provide analgesia. Control animals treated with saline or any of the lidocaine doses exhibited no change in withdrawal latencies between Day 0 and Day 3. In a separate group of ligated animals, lidocaine infusion (0.67 mg/kg/h) that was started 24 h after sciatic ligation surgery reversed the already present thermal hyperalgesia. Average plasma lidocaine concentrations were 0.11, 0.36, and 0.45 &mgr;g/ml for animals receiving 0.33, 0.67 and 1.3 mg/kg/h of lidocaine, respectively. These results suggest that continual systemic infusion of lidocaine prevents or reverses the development of neuropathic pain following chronic constriction injury. These results add to the increasing body of evidence supporting the therapeutic value of preemptive and post‐operative lidocaine administration for the relief of neuropathic pain.

Responses of neurons in the rat ventrolateral orbital cortex to phasic and tonic nociceptive stimulation

Responses of ventrolateral orbital cortex (VLO) neurons to innocuous touch and pressure, and noxious pinch and cold were examined by extracellular recordings. Eight neurons increased their firing rate in a graded fashion to all stimuli applied, 4 units decreased their discharges, and 6 neurons remained unaffected. All responsive units displayed the greatest change in firing rate during noxious cold, and all exhibited whole-body receptive fields. These data further implicate the VLO in nonciception.

Long-term changes in sciatic-evoked A-fiber dorsal horn field potentials accompany loose ligation of the sciatic nerve in rats

Abstract The goal of the present study was to examine whether loose ligation of the sciatic nerve was associated with long‐term changes in neuronal excitability in the spinal dorsal horn in urethane‐anesthetized rats. The sciatic nerve was stimulated with 0.1 ms long pulses at 1 stimulus/5 min, and the evoked dorsal horn field potentials remained stable in the absence of tetanic stimulation. In one set of control and ligated animals, high‐frequency tetanic stimulation was applied to the nerve at 50 Hz (one 400 ms train of twenty 0.1 ms pulses), and the field potentials were recorded again (1 stimulus/5 min) for up to 4 h post‐tetanus. In control animals, this protocol produced significant increases in field potential amplitudes at 15, 30 and 60 min post‐tetanus. Interestingly, after this time the evoked field potentials began to decrease, and attained less than 50% of their pre‐tetanic values at 240 min post‐tetanus. In contrast, in ligated rats the pattern of post‐tetanic potentiation was significantly different as the increases in amplitude persisted, and at 240 min post‐tetanus the field potentials were almost twice their baseline values. In another set of control and ligated animals, low‐frequency tetanic stimulation was applied at 5 Hz (one 400 ms train of two 0.1 ms pulses). Again a differential pattern of post‐tetanic responses between control and ligated rats was seen. In control animals, a significant decrease in amplitude was evident within 30 min, and the depression became progressively more pronounced as the field potentials attained about a quarter of their baseline values at 180 min, and remained at these low levels at 240 min post‐tetanus. On the other hand, in ligated animals, the depression was not significant, and at 240 min post‐tetanus the field potentials were still at about 80% of their baseline values. These data demonstrate that long‐term changes in spinal dorsal horn neuronal excitability accompany sciatic ligation to perhaps contribute to the development of neuropathic pain. These changes may result from a lessening of normally strong inhibitory processes in the spinal dorsal horn to generate conditions which favor post‐tetanic potentiation over depression of dorsal horn neuronal responses.

Muscimol prevents long-lasting potentiation of dorsal horn field potentials in rats with chronic constriction injury exhibiting decreased levels of the GABA transporter GAT-1

The inhibitory activity of gamma‐aminobutyric acid (GABA) is considered critical in setting the conditions for synaptic plasticity, and many studies support an important role of GABA in the suppression of nociceptive transmission in the dorsal horn. Consequently, any injury‐induced modification of the GABA action has the potential to critically modify spinal synaptic plasticity. We have previously reported that chronic constriction injury of the sciatic nerve was accompanied by long‐lasting potentiation of superficial spinal dorsal horn field potentials following high‐frequency tetanus. In this study we examined whether the GABA‐A receptor agonist muscimol would modify post‐tetanic responses in rats with chronic constriction injury. In animals exhibiting maximal thermal hyperalgesia as one sign of neuropathic pain 7 days after loose ligation of the sciatic nerve, spinal application of muscimol (5, 10 or 20 &mgr;g) before the high‐frequency (50 Hz) tetanus produced a long‐lasting depression (rather than potentiation) of spinal dorsal horn field potentials. In separate but related Western immunoblot experiments, we also established that the chronic constriction injury was accompanied by significant decreases in the content of the GABA transporter GAT‐1. These data demonstrated that GABA‐A receptor agonists may effectively influence the expression of long‐lasting synaptic plasticity in the spinal dorsal horn, and that an injury‐induced loss in GABA transporter content may have contributed to a depletion of GABA from its terminals within the spinal dorsal horn. These data lent further support to the notion that the loss of GABA inhibition may have important consequences for the development of neuropathic pain.