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The aim of the present study was to investigate the protein levels of BDNF and IL-1ß in the lumbar dorsal horn of the rat by Western blot analysis following a noxious thermal hind paw stimulation. Ten min, 1h and 3h after the combined chemical and thermal stimulation an up to 2-fold increase in BDNF and Il-1ß protein expression was observed in the lumbar dorsal spinal cord. A pretreatment with the opioid analgesic morphine or the glial cell activation inhibitor minocycline partly attenuated protein expression. The present findings indicate that the BDNF and IL-1ß induction within the dorsal horn may be linked to the development of hyperalgesia, and that opioid analgesics and probably inhibitors of glial cell activation can prevent sensitization in the pain pathway at spinal level., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The responses of sensory neuropeptides during unilateral, Freund's adjuvant-induced, paw inflammation in the rat were examined. After five days of inflammation, the substance P and calcitonin gene-related peptide content in the sciatic nerve supplying the inflamed paw were increased by 60-75% when compared with the contralateral side. At this time-point, there was also a 30-40% increase in the substance P and calcitonin gene-related peptide content of the dorsal root ganglia (L4-L6), and a 40% increase in the calcitonin gene-related peptide content of the L4-L6 segments of the dorsal spinal cord on the inflammation side. In the dorsal root ganglia, calcitonin gene-related peptide content was also increased as early as 12 h and 48 h after induction of paw inflammation. On day 5 of inflammation, the axonal transport of both sensory neuropeptides towards the inflamed paw, as determined after sciatic nerve ligation, was also markedly increased as compared with the control side. Despite this increased transport, the amount of substance P and calcitonin gene-related peptide present in the inflamed paw itself was either reduced or remained unchanged from day 1 through to day 5 of inflammation pointing towards reduced storage and increased release of the peptides in the inflamed tissue. Nerve growth factor content was markedly increased in the sciatic nerve of the inflamed paw with a peak of +136% at time-point 24 h after induction of inflammation. When rats were systemically treated with anti-nerve growth factor serum, the increase in neuropeptide content in the sciatic nerve of the inflamed paw (day 5) was prevented. On the other hand, local injections of nerve growth factor for 5 days into a noninflamed paw were able to induce an increase in substance P and calcitonin gene-related peptide content in the supplying sciatic nerve. These findings point towards a regulatory function for nerve growth factor in vivo in the stimulation of sensory neuropeptide synthesis during prolonged inflammatory processes.

The capsaicin-evoked release of calcitonin gene-related peptide (CGRP) from rat superfused dorsal spinal cord slices was investigated during sustained capsaicin exposure thought to represent equilibrium conditions. The dose-effect relationship for total peptide release evoked by single capsaicin doses (26 min exposure) was very steep with a threshold at 0.06 microM and a maximum at 0.3 microM capsaicin. With concentrations of capsaicin within this range the slow decline of the peptide release in the presence of capsaicin was not a consequence of exhaustion of an available peptide pool nor of neuronal impairment because potassium depolarization was still able to release CGRP. In contrast, with concentrations of capsaicin at 1.5 microM and above, there was a much faster decay of the release after the peak, most probably due to a loss of the secretion capacity caused by neuronal inactivation. When cumulative dose regimens for capsaicin were employed, release of CGRP could be stimulated only up to a dose of 1-1.5 microM capsaicin; further increase in capsaicin concentration was ineffective. This was also most probably due to a loss of the secretion capacity caused by neuronal inactivation and not caused by depletion of a releaseable peptide pool. Release of CGRP evoked by capsaicin concentrations in the range of 0.1-0.3 microM in either dosage protocol was reduced in the presence of Ruthenium Red (RR, 2.5 microM). RR did not reduce neuropeptide release evoked by capsaicin concentrations at or above 1-1.5 microM, nor did it affect the inactivation of the release process at such high capsaicin concentrations. The results demonstrate that, upon sustained exposure to capsaicin, different ranges of concentration can be established at which either only stimulatory or a mixture of stimulatory and inhibitory effects determine the amount of neuropeptides released.

A release of endogenous glutamate (Glu), aspartate (Asp) and gamma-aminobutyric acid (GABA) from rat dorsal spinal cord slices was evoked by a 60 mM K(+)-depolarization (calcium dependent) and by 50 microM veratridine (tetrodotoxin sensitive) indicating a neuronal exocytosis mechanism. No release of the 3 amino acids investigated was obtained with the selective C-fiber excitant capsaicin (1 microM) and after impairment of afferent C-fibers the amount of amino acids released by K(+)-depolarization or veratridine was not reduced. These results suggest that amino acids may be exclusively transmitters of capsaicin-insensitive nerve structures in the rat dorsal spinal cord.

Lofentanil, a very potent and long-acting opiate agonist, was used to evaluate the opioid control of substance P release from primary afferents. Substance P release from the central terminals of primary afferents was studied in the superfused isolated dorsal half of the rat spinal cord. Substance P release as initiated by electrical field stimulation and by capsaicin was found to be diminished by 50% by lofentanil (1 microM) in a naloxone-reversible manner. Substance P release from peripheral terminals of primary afferents was induced by antidromic saphenous nerve stimulation. Release was measured indirectly by its effect on blood flow and plasma extravasation in the rat hind paw. Both antidromic vasodilatation and plasma extravasation were dose dependently inhibited by lofentanil. The inhibition of antidromic vasodilatation by 10 micrograms X kg-1 lofentanil i.p. was completely prevented by 1 mg X kg-1 naloxone. On the other hand, vasodilatation and plasma extravasation induced by infusion of 3.7 pmol X min-1 substance P remained unaffected by lofentanil. It is concluded that lofentanil inhibits the release of substance P from central as well as peripheral terminals of substance P-containing primary afferent neurons.

The aim of the present study was to investigate whether the phosphorylation of ERK1/2 in the rat lumbar dorsal horn and in the parvocellularis part of the paraventricular nucleus can be used to visualize neuronal activity. pERK1/2 fluorescence-immunohistochemistry is specifically suited to mirror neuronal activity in the pain pathway following an acute noxious stimulation. The rat hind paw was either stimulated by noxious heat or by a sequence of mustard oil and noxious heat. Two and 10 min after the thermal stimulation a 3–4-fold increase in cells with pERK1/2 immunoreactivity was observed in lamina I/II of the L3-L5 dorsal horn. The combination of mustard oil with heat led to a 5–6-fold increase in the pERK1/2 signal. The pERK1/2 immunoreactivity in the parvocellularis part of the paraventricular nucleus increased by 2-fold following the heat stimulus, with no further increase following the sequential mustard oil and heat stimulus. A pretreatment with the opioid analgesic morphine or the NMDA antagonist MK-801 markedly attenuated ERK1/2 phosphorylation in both areas of the pain pathway. The present findings support the concept that the pERK1/2 immunofluorescence signal can be used as a quantitative marker for sensitization or inhibition in the pain pathway at spinal and hypothalamic level. [ABSTRACT FROM AUTHOR]