A new paradigm to understand and treat diabetic neuropathy.

The clinical symptoms of diabetic neuropathy (DN) manifest in a time dependent manner as a positive symptoms (i. e. pain, hypersensitivity, tingling, cramps, cold feet etc.) during its early stages and by a loss of function (i. e. loss of sensory perception, delayed wound healing etc.) predominating in the later stages. Elevated blood glucose alone cannot explain the development and progression of DN and the lowering of blood glucose is insufficient in preventing and/or reversing neuropathy in patients with type 2 diabetes. Recently it has been shown that the endogenous reactive metabolite methylglyoxal (MG) can contribute to the gain of function via post-translational modification in DN of neuronal ion channels involved in chemosensing and action potential generation in nociceptive nerve endings. Dicarbonyls, such as MG, that are elevated in diabetic patients, modify DNA as well as extra- and intracellular proteins, leading to the formation of advanced glycation endproducts (AGEs). Increased formation of AGEs leads to increased cellular stress, dysfunction and ultimately cell death. The interaction of AGE-modified proteins through cell surface receptors, such as RAGE, can lead to increased cellular activation and sustained inflammatory responses, which are the molecular hallmarks of the later, degenerative, stages of DN. The direct and indirect effects of dicarbonyls on nerves or neuronal microvascular network provides a unifying mechanism for the development and progression of DN. Targeting the accumulation of MG and/or prevention of RAGE interactions may therefore provide new, more effective, therapeutic approaches for the treatment of DN.

TRPV1, TRPA1, and CB1 in the isolated vagus nerve--axonal chemosensitivity and control of neuropeptide release.

Vagal sensory afferents innervating airways and abdominal tissues express TRPV1 and TRPA1, two depolarizing calcium permeable ion channels playing a major role in sensing environmental irritants and endogenous metabolites which cause neuropeptide release and neurogenic inflammation. Here we have studied axonal chemosensitivity and control of neuropeptide release from the isolated rat and mouse vagus nerve by using prototypical agonists of these transduction channels - capsaicin, mustard oil and the specific endogenous activators, anandamide (methyl arachidonyl ethanolamide, mAEA), and acrolein, respectively. Capsaicin evoked iCGRP release from the rat vagus nerve with an EC50 of 0.12 µM. Co-application of mAEA had a dual effect: nanomolar concentrations of mAEA (0.01 µM) significantly reduced capsaicin-evoked iCGRP release while concentrations ≥ 1 µM mAEA had sensitizing effects. Only 100 µM mAEA directly augmented iCGRP release by itself. In the mouse, 310 µM mAEA increased release in wildtype and TRPA1-/- mice which could be inhibited by capsazepine (10 µM) and was completely absent in TRPV1-/- mice. CB1-/- and CB1/CB2 double -/- mice equally displayed increased sensitivity to mAEA (100 µM) and a sensitizing effect to capsaicin, in contrast to wildtypes. Acrolein and mustard oil (MO)--at µM concentrations--induced a TRPA1-dependent iCGRP release; however, millimolar concentrations of mustard oil (>1mM) evoked iCGRP release by activating TRPV1, confirming recent evidence for TRPV1 agonism of high mustard oil concentrations. Taken together, we present evidence for functional expression of excitatory TRPV1, TRPA1, and inhibitory CB1 receptors along the sensory fibers of the vagus nerve which lend pathophysiological relevance to the axonal membrane and the control of neuropeptide release that may become important in cases of inflammation or neuropathy. Sensitization and possible ectopic discharge may contribute to the development of autonomic dysregulation in visceral tissues that are innervated by the vagus nerve.

Sensitized peripheral nociception in experimental diabetes of the rat.

Painful neuropathy is a common complication of diabetes. Particularly in the early stage of diabetic neuropathy, patients are characterized by burning feet, hyperalgesia to heat, and mechanical stimuli, as if residual nociceptors were sensitized. Such symptoms are barely explained by common pathophysiological concepts of diabetic neuropathy. Diabetes was induced in Wistar rats by streptozotocin (STZ). After 4 weeks behavioral testing (Plantar test, Randall-Selitto) was conducted. Basal and stimulated release of calcitonin gene-related peptide (CGRP), Substance P (SP) and prostaglandin E(2) (PGE(2)) from isolated skin and sciatic nerve were assessed by enzyme immunoassays. Electrophysiological properties of identified nociceptors under hyperglycemic, hypoxic, and acidotic conditions were investigated using the skin-nerve preparation. The diabetic rats showed hyperalgesia to heat and pressure stimulation. The basal CGRP/SP release was reduced, but chemical stimulation with bradykinin induced greater release of SP, CGRP and PGE(2) than in control animals. In contrast, capsaicin-stimulated CGRP release was reduced in sciatic nerves. Hypoxia per se lowered von Frey thresholds of most C-nociceptors to half. Hyperglycemic hypoxia induced ongoing discharge in all diabetic but not control C-fibers which was further enhanced under acidosis. Sensory and neurosecretory nociceptor functions are sensitized in diabetes. Diabetic C-fibers show exaggerated sensitivity to hyperglycemic hypoxia with and without additional acidosis, conditions that are thought to mimic ischemic episodes in diabetic nerves. Ongoing C-fiber discharge is known to induce spinal sensitization. Together with altered receptor and ion channel expressions this may contribute to painful episodes in diabetic neuropathy.

Projected pain from noxious heat stimulation of an exposed peripheral nerve--a case report.

Distinct sensory properties of unmyelinated axons in the isolated rat sciatic nerve have previously been revealed by measuring stimulated neuropeptide (CGRP) release in response to noxious stimuli. Axonal sensitization to heat by inflammatory mediators has been demonstrated and shown to depend on the heat- and proton-activated ion channel TRPV1. Recently, we have demonstrated in vitro that heat stimulation of nociceptive axons generates ectopic action potential discharge which resembles the heat response of the corresponding cutaneous nerve endings. It remained however, to be established whether adequate axonal stimulation could also generate projected sensations in a conscious human subject. In a singular human trial, the superficial radial nerve (SR) was exposed and stimulated mechanically as well as with noxious cold (3 degrees C). These stimuli were unable to induce any conscious local or projected sensations. However, controlled radiant heat applied to the nerve resulted in intense slowly adapting burning pain sensations projected into the center of the SR innervation area. No local sensation was reported. Thus, presumably activated nervi nevorum in the sheath of a healthy nerve do not cause conscious sensations, while axons of passage in mid-nerve exhibit a sensory transduction capacity for noxious heat though not for mechanical and cold stimulation. Axonal heat transduction may therefore become a source of ectopic discharge and neuropathic pain when heat threshold drops to body temperature as is the case with peripheral nerve endings in inflamed skin.

Mechanisms of potassium- and capsaicin-induced axonal calcitonin gene-related peptide release: involvement of L- and T-type calcium channels and TRPV1 but not sodium channels.

We have previously shown that capsaicin, noxious heat, protons and potassium ions (K(+)) induce a graded, calcium- and receptor-dependent increase of immunoreactive calcitonin gene-related peptide (iCGRP) release from isolated rat sciatic axons. Morphological evidence for axonal vesicular exocytosis has also been presented. Here we determine the differential contribution of voltage-gated calcium and sodium channels to high extracellular potassium and capsaicin-induced iCGRP secretion. Blockade of L-type calcium channels significantly decreased the K(+)-induced axonal response (nimodipine (10 microM) by 66% and methoxyverapamil, D600 (50 microM), by 77%). Interestingly, however, D600 was unable to reduce the capsaicin-induced iCGRP release. Omega-Conotoxin GVIA (1 microM), a N-type blocker, and omega-agatoxin TK (0.1 microM), a P/Q-type blocker, had no significant effect. Also the anticonvulsant gabapentin (50 microM and 100 microM), reported to impede calcium channels, was ineffective. Inhibition of low threshold T-type calcium channels by mibefradil (10 microM) significantly reduced potassium (by 47%) but not capsaicin-stimulated iCGRP release. Reduction of total sodium channel conductance by tetrodotoxin (1 microM), lidocaine (10 microM, 50 microM or 500 microM) or by replacement of extracellular sodium with choline-chloride did not result in a reduction of either potassium- or capsaicin-induced axonal iCGRP release. These results suggest that slow depolarization by high extracellular potassium activates axonal low threshold (T-type) as well as high threshold-activated (L-type) voltage-gated calcium channels to mediate iCGRP release, and that capsaicin-induced release is largely dependent on calcium influx through TRPV1. Action potential generation and propagation are not required for axonal release mechanisms.

Can receptor potentials be detected with threshold tracking in rat cutaneous nociceptive terminals?

Threshold tracking of individual polymodal C- and Adelta-fiber terminals was used to assess membrane potential changes induced by de- or hyperpolarizing stimuli in the isolated rat skin-nerve preparation. Constant current pulses were delivered (1 Hz) through a tungsten microelectrode inserted in the receptive field, and the current amplitude was controlled by feedback with a laboratory computer programmed to serially determine the electrical threshold using the method of limits. During threshold tracking, the receptive fields of the fibers were heated (32-46 degrees C in 210 s) or superfused with modified synthetic interstitial fluid containing either 0, 20, 40, 50, or 60 mM [K+], phosphate buffer to pH 5.2 or 6.1, or bradykinin (BK, 10(-8)-10(-5) M). High [K+]e decreased the current threshold for activation by 6-14% over 120 s, whereas K+-free superfusion augmented the threshold by >5%, and after some delay, also induced ongoing discharge in 60% of units. pH 6.1 and 5.2 caused an increase in threshold of 6 and 18%, respectively, and 30% of the fibers were excited by low pH, although the change in threshold of pH responsive and unresponsive fibers did not differ significantly, suggesting a general excitability decrease induced by protons. Heat stimulation increased the mean threshold and conduction velocity of the fibers tested and resulted in activity in 78% of units. Additionally, for these units, activation was preceded by a significant decrease in threshold compared with the tracked thresholds of fibers unresponsive to heat. Bradykinin also led to a significant threshold decrease before activation. In conclusion, the technique of threshold tracking proved suitable to assess changes in excitability resulting from receptor currents evoked by noxious heat and bradykinin in the terminal arborization of cutaneous nociceptors.

Morphological evidence for functional capsaicin receptor expression and calcitonin gene-related peptide exocytosis in isolated peripheral nerve axons of the mouse.

Rat sciatic nerve axons express capsaicin, proton and heat sensitivity and respond to stimulation with a Ca2+-dependent and graded calcitonin gene-related peptide (CGRP) release. In this study we demonstrate that similar functions, including capsaicin-induced CGRP release, are to be found in the desheathed sciatic nerve of the mouse. We have morphologically investigated the mechanisms of this axonal release in regions away from the active zones of synapses. Capsaicin receptor 1 (TRPV1) and CGRP immunostaining was performed using electron microscopic visualization. TRPV1 was identified in the axoplasm and inside vesicles--presumably on axonal transport--as well as in considerable quantity in the axonal plasma membrane of unmyelinated nerve fibers. Most of the unmyelinated axons were immunopositive for CGRP and in unstimulated nerves CGRP-containing vesicles almost entirely filled the axoplasm. After capsaicin stimulation (10(-6) M for 5 min), the fibers appeared depleted of CGRP with only few vesicles remaining as well as some residual staining of the axoplasm. In addition a large number of vesicles were fused with the axonal membrane, forming classical exocytotic figures--the omega structures--lined with CGRP immunoreactive product. These results present morphological evidence for the distribution of TRPV1 along unmyelinated axons in peripheral nerve and also provide the first demonstration of vesicular neuropeptide exocytosis along unmyelinated axons in peripheral nerve.

Responsiveness of C-fiber nociceptors to punctate force-controlled stimuli in isolated rat skin: lack of modulation by inflammatory mediators and flurbiprofen.

Although cutaneous C-fiber nociceptors show dramatic inflammatory sensitization to heat, they do not appear to get sensitized to the mechanical stimulation by von Frey hairs. We employed force-controlled punctate electromechanical stimulation to receptive fields of 61 characterized C-fibers in the isolated rat skin-saphenous nerve preparation. In general: low-in contrast to higher-threshold units showed greater dynamic sensitivity and response magnitude, an earlier onset and a stronger degree of adaptation, the latter due to the linear rise of the force stimulus. On this methodological basis three groups of units were subject to a mix of inflammatory mediators, to flurbiprofen or to control solution. Subsequent mechanostimulation revealed a good reproducibility of the control response and no significant changes in the treatment groups. In conclusion, even refined mechanostimulation was unable to demonstrate sensitization of the predominant nociceptor classes in the rat skin.

Noxious heat-induced CGRP release from rat sciatic nerve axons in vitro.

Noxious heat may act as an endogenous activator of the ionotropic capsaicin receptor (VR1) and of its recently found homologue VRL1, expressed in rat dorsal root ganglion cells and present along their nerve fibres. We have previously reported that capsaicin induces receptor-mediated and Ca++-dependent calcitonin gene-related peptide (CGRP) release from axons of the isolated rat sciatic nerve. Here we extended the investigation to noxious heat stimulation and the transduction mechanisms involved. Heat stimulation augmented the CGRP release from desheathed sciatic nerves in a log-linear manner with a Q10 of approximately 15 and a threshold between 40 and 42 degrees C. The increases were 1.75-fold at 42 degrees C, 3.8-fold at 45 degrees C and 29.1-fold at 52 degrees C; in Ca++-free solution these heat responses were abolished or reduced by 71 and 92%, respectively. Capsazepine (10 microm) and Ruthenium Red (1 microm) used as capsaicin receptor/channel antagonists did not significantly inhibit the heat-induced release. Pretreatment of the nerves with capsaicin (100 microm for 30 min) caused complete desensitization to 1 microm capsaicin, but a significant heat response remained, indicating that heat sensitivity is not restricted to capsaicin-sensitive fibres. The sciatic nerve axons responded to heat, potassium and capsaicin stimulation with a Ca++-dependent CGRP release. Blockade of the capsaicin receptor/channels had little effect on the heat-induced neuropeptide release. We conclude therefore that other heat-activated ion channels than VR1 and VRL1 in capsaicin-sensitive and -insensitive nerve fibres may cause excitation, axonal Ca++ influx and subsequent CGRP release.

Pro- and anti-inflammatory actions of ricinoleic acid: similarities and differences with capsaicin.

We have investigated the pro- and anti-inflammatory effects of ricinoleic acid (RA), the main active principle of castor oil, in an experimental model of blepharitis induced by intradermal injection of carrageenan in the guinea-pig eyelid and its possible capsaicin-like mode of action on acutely dissociated rat dorsal root ganglia (DRG) neurons in vitro. Topical treatment with RA (10-100 mg/guinea-pig) or capsaicin (1-10 mg/guinea-pig) caused eyelid reddening and oedema. At lower doses (0.3-3 mg/guinea-pig and 0.009-0.09 mg/guinea-pig for RA and capsaicin, respectively) both drugs significantly potentiated the eyelid oedema induced by carrageenan. The tachykinin NK1 receptor antagonist FK 888 (0.59 mg/kg s.c.) abolished the potentiation of carrageenan-induced eyelid oedema induced by either RA or capsaicin. The neutral endopeptidase inhibitor, thiorphan (1.3 mg/kg i.v.) significantly enhanced the potentiation of carrageenan-induced eyelid oedema produced by RA. This potentiating effect was abolished by FK 888. Repeated (8 days) topical application of RA (0.9 mg/guinea-pig) or capsaicin (0.09 mg/guinea-pig) inhibited the carrageenan-induced eyelid oedema. This anti-inflammatory effect was accompanied by a reduction (75%-80% of SP and 46%-51% of NKA) in tachykinin content of the eyelids, as determined by radioimmunoassay. In dissociated rat DRG neurons, RA (0.1 mM for 5 min) significantly inhibited the inward currents induced by application of capsaicin (1 microM) and/or low pH (5.8), without inducing any currents by itself or changing voltage-dependent currents. Moreover, after 24-h incubation, RA (0.1 mM) significantly decreased the capsaicin (1 microM)-induced calcitonin gene-related peptide (CGRP) release from rat DRG neurons, whereas acute drug superfusion did not evoke CGRP release by itself. Summarizing, RA possesses capsaicin-like dual pro-inflammatory and anti-inflammatory properties which are observed upon acute and repeated application, respectively. However, unlike capsaicin, RA does not induce inward current in DRG neurons and it is devoid of algesic properties in vivo.

Muscarinic M2 receptors inhibit heat-induced CGRP release from isolated rat skin.

The action of cholinergic agonists on modulating basal and heat-induced CGRP release was investigated in isolated rat skin. Nicotine (10(-6), 10(-5) and 10(-4) M) induced a bimodal increase of CGRP release, that was significant for the two larger concentrations (by 113 and 36%, respectively). On the contrary, muscarine (10(-4) M) and arecaidine (10(-5) M) significantly decreased the basal CGRP release (by 16 and 23%, respectively). The substantial increase of CGRP release evoked by noxious heat (47 degrees C) remained unaltered upon co-application of nicotine, but was diminished by 35% upon muscarine. Arecaidine was more effective in this respect causing significant dose-dependent depressions by 30% (at 10(-6) M) and by 60% (at 10(-5) M). These data support a role of muscarinic M2 receptors in nociceptor desensitization.

Excitatory nicotinic and desensitizing muscarinic (M2) effects on C-nociceptors in isolated rat skin.

The actions of different cholinergic agonists and antagonists were investigated on nociceptive afferents using the rat skin-saphenous nerve preparation, in vitro. Nicotine was able to weakly excite C-nociceptors and to induce a mild sensitization to heat stimulation (in 77% of tested fibers) in a dose-dependent manner (10(-)6 to 10(-)5 m), but it caused no alteration in mechanical responsiveness tested with von Frey hairs. Muscarine did not induce a significant nociceptor excitation, but almost all fibers exhibited a marked desensitization to mechanical and heat stimuli in a dose-dependent manner (from 10(-)6 to 10(-)4 m). The muscarinic effects could be prevented by the general muscarinic antagonist scopolamine (10(-)5 m), by the M3 antagonist 1,1-dimethyl-4-diphenylacetoxypiperidium oxide (10(-)6 m) co-applied with the M2 antagonist gallamine (10(-)5 m), and by gallamine alone. As positive control we used the relatively M2-selective agonist arecaidine (10(-)6 to 10(-)5 m), obtaining a similar desensitizing effect as with muscarine. Finally, we performed an immunocytochemical study that demonstrated the presence of M2 but not M3 receptors in thin epidermal nerve fibers of the rat hairy skin. Altogether, these data demonstrate opposite effects of nicotinic and muscarinic receptor stimulation on cutaneous nociceptors. M2 receptor-mediated depression of nociceptive responsiveness may convey a therapeutic, i.e., analgesic or antinociceptive, potential.

Denervation and NKI receptor block modulate stimulated CGRP and PGE2 release from rat skin.

We investigated the possible neurogenic origin of prostaglandin E2 (PGE2) in the rat skin, in vitro. The hairy skin of one hindpaw was denervated and one week later the dorsal hindpaws were skinned to study the release of calcitonin gene-related peptide (CGRP) and PGE2 using the EIA technique. Stimulation with bradykinin (BK) caused a significant release of CGRP (1.4-fold increase) and PGE2 (3-fold) which was massively augmented under neurokinin I (NKI) receptor antagonist treatment (CGRP: 4-fold, PGE2: 5-fold). In denervated skin the BK-evoked CGRP release was lost whereas the PGE2 release was unchanged. Thus, neither nerve endings nor neuropeptides contribute essentially to BK-induced PGE2 release in the skin. However, excessive neuropeptide levels, as under NKI blockade facilitate PGE2 formation, which may play a role in sustained inflammation.

Rat peripheral nerve components release calcitonin gene-related peptide and prostaglandin E2 in response to noxious stimuli: evidence that nervi nervorum are nociceptors.

The presence of an intrinsic afferent innervation of nerves and their connective tissues (nervi nervorum) suggests that these neural elements participate in sensation and pathological processes affecting nerves. Primary afferent nociceptors contain and release neuropeptides including calcitonin gene-related peptide, implicated in inflammatory vasodilatation. We sought to evaluate the ability of different peripheral nerve components, in vitro, to release calcitonin gene-related peptide and prostaglandin E2 in response to electrical and noxious chemical stimuli, using sensitive enzyme immunoassays. We observed significant increases in both calcitonin gene-related peptide and prostaglandin E2 in response to a mixture of inflammatory mediators (bradykinin, histamine, and serotonin; 10(-5) M) applied to the intact nerves (+37% and +700%, respectively) and isolated sheaths (35% and 430%, respectively), but not when this mixture was applied to isolated axons. Proximal (antidromic) but not distal (orthodromic) electrical stimulation also evoked a comparable release of calcitonin gene-related peptide (+30%) from intact nerves. These results suggest that nervi nervorum nociceptors participate in neural inflammation. Capsaicin (10(-6) M) elicited a very large release of calcitonin gene-related peptide when applied to either the intact nerve (+400%), isolated sheaths (+500%), or isolated axons (1400%). The latter effect was substantially but not completely blocked by Ruthenium Red and capsazepine, and was completely blocked using a calcium-free bathing solution. The results support the presence of capsaicin receptors in peripheral nerves that can effect calcitonin gene-related peptide release from axons as well as from terminals.

Stimulated prostaglandin E2 release from rat skin, in vitro.

The excitatory effect of bradykinin (BK) and of low pH on nociceptors appears to partly depend on secondary release of prostaglandins from the surrounding tissue. Rat skin, in vitro, is introduced as a novel model to measure basal and stimulated release of PGE2 and, in future, other substances relevant to nociception, such as neuropeptides. Flaps of hairy skin (n=57) from the rat saphenous region of the hindpaw were subcutaneously excised and fixed on acrylic rods, the corium side exposed. The preparations were equilibrated in carbogen gassed "synthetic interstitial fluid" (SIF) for 30 minutes. The skin flaps were then immersed for 5 minutes each in 9 consecutive glass tubes, which were mounted in a shaking bath at 32 degrees C. Each tube was filled with 5 ml of gassed SIF, the third tube contained inflammatory mediator(s) dissolved in SIF or solutions of low pH. After passage of the skin flap, the eluates were deep frozen (-70 degrees C) and the PGE2 content measured, off-line, using an enzyme immuno-assay. As stimulants, BK at 10(-5) M (n=9) and 10(-6) M (n=4) and BK in equimolar combination with histamine (HA) and serotonin (5-HT; 10(-5) M: n=8, 10(-6) M: n=6, 10(-7) M: n=6) dose-dependently increased PGE2 release. Considering the total amount of PGE2 secreted the combination of inflammatory mediators caused a significantly greater release of PGE2 at 10(-5) and 10(-6) M (p<0.01, Kruskal-Wallis test) than BK stimulation alone. Racemic flurbiprofen caused a profound depression of basal and stimulated release. Solutions of high proton concentration are known to stimulate and sensitize nociceptors. However, phosphate buffered SIF at pH 6.1 and 6.4 caused a substantial and significant decrease of the PGE2 release, probably due to low-pH block of phospholipases. Thus, algogenic potency of mediators does not necessarily match their pro-inflammatory action.