Disparate changes in the expression of transient receptor potential vanilloid type 1 receptor mRNA and protein in dorsal root ganglion neurons following local capsaicin treatment of the sciatic nerve in the rat.

In situ hybridization, quantitative reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis were applied to study the changes in expression of the major nociceptive ion channel transient receptor potential vanilloid type 1 receptor (TRPV1) after the perineural application of capsaicin or nerve transection. In control rats, quantitative morphometric and statistical analyses of TRPV1 protein and mRNA expression in L5 dorsal root ganglion cells revealed distinct populations of small (type C) and small-to-medium (type B) neurons, which showed very high and moderate levels of TRPV1, whereas larger (type A) neurons mostly did not express this receptor. After either transection or capsaicin treatment of the sciatic nerve, immunohistochemistry and Western blotting demonstrated a massive (up to 80%) decrease in the proportion of TRPV1-immunoreactive neurons and TRPV1 protein at all postoperative survival times. In situ hybridization indicated marked decreases (up to 85%) in the proportion of neurons that expressed TRPV1 mRNA after sciatic nerve transection. In contrast, although perineural treatment with capsaicin resulted in similar substantial decreases in the proportions of type B and C neurons of the L5 dorsal root ganglia 3 days postoperatively, a clear-cut tendency to recovery was observed thereafter. Hence, the proportions of both type B and C neurons expressing TRPV1 mRNA reached up to 70% of the control levels at 30 days postoperatively. In accord with these findings, quantitative RT-PCR revealed a marked and significant recovery in TRPV1 mRNA after perineural capsaicin but not after nerve transection. These observations suggest the involvement of distinct cellular mechanisms in the regulation of the TRPV1 mRNA expression of damaged neurons, specifically triggered by the nature of the injury. The present findings imply that the antinociceptive and anti-inflammatory effects of perineurally applied capsaicin involve distinct changes in neuronal TRPV1 mRNA expression and long-lasting alterations in (post)translational regulation.

Galanin mediated inhibitory nervous modulation of cutaneous vascular reactions.

Noxious stimulation induces local inflammatory responses in a variety of mammals but these reactions are only faint in avian species. The possibility that endogenous galanin inhibits neurogenic vascular responses in avians was tested in the wing skin of anaesthetized pigeons. Intraarterial infusion of nanomolar concentrations of the specific galanin antagonist M35 dose dependently enhanced the small mustard oil induced increase of skin blood flow measured by means of a Laser Doppler Imager. Similarly, the small transient vasodilatation following electrical stimulation of a cutaneous nerve was also enhanced by M35. The effect of M35 was not observed after chronic denervation. Coperfusion of M35 dose dependently augmented the histamine and bradykinin induced plasma extravasation revealed by skin microdialyses, but this effect was abolished in the chronically denervated skin. However, chronic denervation per se enhanced the plasma extravasation induced by histamine but not by bradykinin and this effect was diminished by coperfusion of galanin. The results suggest an inhibitory modulation of cutaneous neurogenic inflammatory reactions by endogenous galanin in the pigeon.

Evidence for an inhibition by endogenous galanin of neurogenic cutaneous vasodilatation in the pigeon.

The effect of high affinity galanin antagonist M35 on neurogenic cutaneous vasodilatation has been studied in the pigeon using a Laser Doppler Imager. Cutaneous application of mustard oil or antidromic electrical stimulation of a cutaneous nerve produced a small increase in skin blood flow. Close arterial injection of M35 prior to chemical or electrical stimulation resulted in a marked augmentation of the vasodilatory response. This effect was abolished by chronic denervation. The results suggest a nerve-mediated inhibitory effect of endogenous galanin on neurogenic cutaneous vasodilatation in the pigeon skin and provide the first experimental evidence for an inhibitory local regulatory function of cutaneous sensory nerves at least in the avian skin.

Inhibition of the neurogenic inflammatory response by lidocaine in rat skin.

Axon reflex vasodilatation and neurogenic plasma extravasation are characteristic cutaneous vascular responses mediated by neuropeptides released from stimulated capsaicin-sensitive sensory nerve endings. Intracutaneous injections of local anaesthetics inhibit the axon-reflex flare elicited by chemical irritants in human skin. Results of earlier reports on the effects of local anaesthetics on neurogenic plasma extravasation are controversial. The aim of the present study, therefore, was to re-examine the effect of the local anaesthetic lidocaine on the neurogenic inflammatory response of rat skin. The effects of lidocaine on cutaneous inflammatory reactions were measured quantitatively by means of the Evans blue technique. Intracutaneous injection of lidocaine resulted in a dose-dependent inhibition of the neurogenic inflammation elicited by mustard oil and of the dye leakage response to compound 48/80 or histamine. It is suggested that the site of this inhibition is beyond the sensory nerve terminal, presumably at the level of the vascular endothelium.

Capsaicin treatment induces selective sensory degeneration and increased sympathetic innervation in the rat ureter.

Quantitative immunohistochemistry was used to study the innervation of the ureter in adult rats pretreated with capsaicin as neonates (50 mg/kg) or as adults (100-150 mg/kg, 10-22 days prior to being killed) using antibodies against protein gene-product 9.5, neuron-specific enolase, substance P, calcitonin gene-related peptide, neuropeptide Y, dopamine-beta-hydroxylase and vasoactive intestinal polypeptide. The number of calcitonin gene-related peptide- and substance P-containing fibres was reduced in the subepithelial plexus (adult capsaicin treatment < 1%, neonatal treatment < 5% of control), the submucosa (adult treatment < 11%; neonatal treatment < 51%) and in the smooth muscle layer and adventitia (adult treatment < 11%; neonatal treatment < 58%). Fibres immunoreactive for protein gene-product 9.5, a general neuronal marker, were reduced to 11% (adult treatment) or 0.5% (neonatal treatment) in the subepithelial plexus, but unchanged in the other layers, indicating a selective regional degeneration. In the smooth muscle layer the number of neuropeptide Y- and vasoactive intestinal polypeptide-containing nerve fibres was not altered by capsaicin. The number of neuropeptide Y fibres in the subepithelial plexus, however, was significantly increased after adult treatment (174% of control). After neonatal capsaicin treatment the intensity of the neuropeptide Y immunoreactivity was increased, more neuropeptide Y-positive nerve bundles were found and immunoreactive cell bodies were observed regularly in the adventitia of the ureter. The data indicate that capsaicin produces a selective degeneration of most afferent fibres in the subepithelial plexus of the rat ureter. This loss of capsaicin-sensitive afferent nerves evokes neuroplastic changes resulting in a hyperinnervation by neuropeptide Y-immunoreactive, presumably sympathetic fibres. The results suggest a mutual regulation of the pattern and density of innervation of peripheral target tissues by sensory and sympathetic neurons.

Reduction of substance P binding sites in the spinal dorsal horn after perineural capsaicin treatment in the rat.

The long-term effect of perineural capsaicin treatment on the distribution of substance P (SP) binding sites was studied in the rat spinal dorsal horn using 125I-labelled Bolton-Hunter-SP. Three months after local application of capsaicin onto the sciatic nerve quantitative evaluation of the autoradiograms revealed a significant decrease in the density of SP binding sites of up to 48% in regions of laminae I and II of the spinal dorsal horn somatotopically related to the capsaicin treated sciatic nerve. It is suggested that reduction in SP binding may result from transganglionic and transsynaptic degenerative changes affecting postsynaptic structures. Changes in the distribution of SP binding sites may significantly contribute to functional alterations observed after perineural treatment with capsaicin.

Mustard oil-induced cutaneous inflammation in the pig.

Recent findings indicate that chemical stimulation of the porcine skin with capsaicin evokes a flare response similar to that observed in man. The aim of the present study was to elucidate whether chemical stimulation of cutaneous capsaicin-sensitive nerve endings with mustard oil produces neurogenic inflammatory reactions in the pig. The application of mustard oil onto the abdominal skin of domestic pigs resulted in a pronounced flare response. After a previous intravenous injection of a solution of Evans blue, the skin area in contact with the irritant turned dark blue, indicating a marked extravasation of albumin. Quantitative estimation of the dye content of the skin supported this conclusion. The technique of vascular labelling revealed a delicate network of small subepidermal blood vessels in histological preparations after the application of mustard oil following a previous intravenous injection of colloidal silver. Labelled blood vessels were not noted outside the treated area. The present results show that mustard oil produces a strong cutaneous inflammatory response in the pig, and suggest that the porcine skin provides a valuable model for study of the significance of capsaicin-sensitive sensory nerves in vascular and other cutaneous reactions.

Altered distribution of dorsal root fibers in the rat following neonatal capsaicin treatment.

Distribution of primary afferent fibers was studied in intact and neonatally capsaicin treated rats by the application of horseradish peroxidase to the central branch of the transected lumbar dorsal roots. Coarse primary afferent fibers entered the spinal cord through the larger medial portion of the rootlet and arborized in the deeper part of the dorsal horn (laminae III and IV). Fine fibers reached the spinal cord through the smaller lateral portion of the rootlet and arborized in the superficial portion of the dorsal horn (lamina I and outer portion of lamina II). The technique used was inadequate to stain fine, unmyelinated primary afferent fibers terminating in the larger inner portion of lamina II. After neonatal capsaicin treatment (50 mg/kg) the flame-shaped arborizations of thick primary afferent fibers terminating in intact rat in laminae III and IV spread dorsally and occupied the inner portion of lamina II in the larger lateral sector of the dorsal horn. Medially the dense arborization of a different type of thick primary afferent fibers sprouted up to the white-gray border. The border between the lateral and medial sector was sharp and only slightly varied in localization from experiment to experiment. The sprouting fibers established complicated synaptic contacts with dendrites and axon terminals. The rearrangement of primary afferent fibers after neonatal capsaicin treatment confirmed earlier results and revealed a mediolateral difference in the fiber organization of the dorsal horn indicating differences in the projection from hairy vs non-hairy skin areas.

Capsaicin prevents histamine-induced itching.

The effects of topical treatment with capsaicin or mustard oil on histamine-induced pruritus, wheal formation and flare response were studied in the human skin. Capsaicin pretreatment resulted in a reversible marked reduction or abolition of the axon reflex flare, but did not influence whealing. Itching was also strongly diminished or even abolished, provided that the flare response was completely blocked. The onset of itching was significantly promoted by pretreatment of the skin with mustard oil, inducing axon reflex vasodilatation. It is concluded that, in addition to the axon reflex flare, capsaicin-sensitive peptide-containing primary afferent neurones are also intimately involved in the mediation of the sensation of itching.

Effect of capsaicin on morphine analgesia--possible involvement of hypothalamic structures.

1. Rats were treated with 50 mg/kg capsaicin on the second day of life or at the age of 2--3 months. The effect of morphine on the nociceptive threshold, as determined by the reaction time in tail withdrawal test, was measured 3--4 and 1--2 months after capsaicin pretreatment, respectively. 2. The analgesic effect of morphine was markedly attenuated in rats treated with capsaicin in the adult age, while neonatal capsaicin treatment did not affect morphine analgesia. 3. Pretreatment of adult rats with capsaicin results in the impairment of certain hypothalamic preoptic neurones, while neonatal capsaicin treatment induces selective degeneration of chemosensitive primary sensory neurones without affecting hypothalamic neurones. Therefore, it is suggested that in the analgesic effect of morphine the capsaicin-sensitive neurones of the preoptic area are involved, and the contribution of spinal mechanisms might be of minor importance. Thus, the preoptic region may be an important link in endogenous pain controlling systems.

[Capillary fluorescence in brain smears]. / Capillaris-fluorescencia vizsgálata agyi kenetekben

A histochemical technique for the detection of catecholamines accumulated by endothelial cells of the capillaries of rat brain after L-DOPA treatment is reported. The main point is to obtain smaers from the cut surface of the brain without damage to the capillaries. Catecholamine raction in the latter, then should be examined by the Fakk's technique. The method described is very quick and gives an exact localization of catecholamines accumulated in the wall of the cerebral cpaillaries. It is a useful tool for the investigation of the monoaminoerg brain-blood barriere.