Akio Hiura

Quantitative estimation of the effects of capsaicin on the mouse primary sensory neurons

Studies were made on the quantitative effects of capsaicin on the L4 dorsal roots and their ganglia (DRG) in mice. Capsaicin at a dose of 50 mg/kg was injected s.c. 1-3 times into neonatal mice, while other mice received only vehicle as controls. After capsaicin treatment unmyelinated fibers decreased by 41.3-75.3% compared to controls. After 2 or 3 capsaicin injections there was a greater decrease in unmyelinated fibers than after a single treatment. Capsaicin had most effect on Schwann cell subunit size of more than 13 axons. Reduction of myelinated fibers ranged from 6.1 to 11.6% (mean: 7.9) in 4 mice. A beta and A delta fibers of 4-5 microns diameter were significantly reduced, whereas A delta fibers of less than 2.0 microns diameter were slightly increased. Small DRG neurons (50-500 microns 2 area) decreased by 50.6-77.4% (mean: 69.6) compared to controls. In addition, capsaicin caused a decrease of 14.2-51.6% (mean: 35.2, n = 4) in large-sized neurons (500-1950 microns 2 area). Thus, capsaicin widely affects small and certain types of large neurons as well as unmyelinated and certain myelinated fibers.

Age-related changes in the response to thermal noxious heat and reduction of C-fibers by neonatal treatment with capsaicin.

Developmental changes of the response to nociceptive heat were examined in mice treated with capsaicin (50 mg/kg) on postnatal days 2-15. Tests of hot-plate (55 degrees C) and irradiation by infrared (IR test) were carried out after 10 days of capsaicin administration up to 120 days at intervals of 10 or 20 days. The time until forepaw (hot-plate) and hindpaw (IR test) withdrawal was assessed as the response latencies to suprathreshold and thermal threshold, respectively. Moreover, the numbers of unmyelinated C-fibers and myelinated fibers in the L4 dorsal roots of the animals treated on postnatal days 2 and 5 were counted on electron micrograph montages. Despite the marked reduction of C-fibers (60% mean decrease) in the 4 dorsal roots of the animals treated on postnatal day 2, thresholds were normal compared with those of controls. However, the animals treated with capsaicin on postnatal day 5 showed an apparent delay of thermal threshold latency only in the IR test, although the mean reduction of C-fibers was very likely the same as that of the animals pretreated on day 2. The reduction of C-fibers in mice treated on postnatal days 10 and 15 was lower than the animals treated on days 2 or 5, but their threshold latencies were significantly increased (hypoalgesia). A possible implication of these findings is discussed on the basis of the development of inhibitory systems in the intraspinal and supraspinal dorsal horn and sprouting from the surviving primary afferent neurons in the superficial dorsal horn.

Age-Dependent Attenuation of the Decrease of C Fibers by Capsaicin and Its Effects on Responses to Nociceptive Stimuli

The effects of subcutaneous treatment of mice 10, 15, or 20 days (young) or 30 or 60 days (adult) of age with capsaicin on development of unmyelinated (C) fibers in the L4 dorsal roots were examined. The responses of the mice 2-4 months later to thermal (hot plate; 55 degrees C) and neurogenic plasma extravasative (chemogenic nociception) stimuli were evaluated. Capsaicin treatment 10 days after birth affected development of myelinated fibers significantly (7.9% reduction). Capsaicin treatment 10, 15, 20, and 30 days after birth reduced the number of C fibers 11.0-51.7% and even treatment 60 days after birth caused a significant decrease (10.0%) in the mean number of C fibers. The destruction of C fibers by capsaicin was attenuated during development, but individual differences in the reduction of C fibers were observed on and after 15 days of life and seemed to become more marked over time. Neurogenic plasma extravasation related to primary sensory neurons was decreased by capsaicin, irrespective of the time of treatment. In parallel with reduction of C fibers, hot-plate latency was increased significantly by treatment of young animals with capsaicin. These results suggest that the effect of capsaicin on thermal nociception is age-dependent and is correlated with decrease of C fibers. However, a marked increase in hot-plate latency did not always correspond to a marked decrease of C fibers. In contrast, the reduction of plasma extravasation was not age-dependent: Reduced extravasation of dye persisted during development.(ABSTRACT TRUNCATED AT 250 WORDS)

Is thermal nociception only sensed by the capsaicin receptor, TRPV1?

Mammalian heat pain perception is well documented as a molecular event in the primary afferent neurons expressing TRPV1. Six types of thermo-TRPs were found, i.e., TRPV1–4, TRPM8 and TRPA1. The former TRPV1, 2 and TRPV3, 4 are sensitive to noxious heat and warmth, and the latter two are sensitive to cool or cold, respectively. We attempted to provide a hypothesis to explain the paradox in which TRPV1 knockout mice and capsaicin-pretreated mice with severe loss of small dorsal root ganglion (DRG) neurons behave normally to noxious heat. From the general view that TRPV1 is preferentially expressed in C-fibers responding to a moderate thermal threshold (>43°C) and TRPV2 in Aδ-fibers to high threshold temperatures (>52°C), the above phenomenon is perplexing. Woodbury et al. (J Neurosci 24:6410–6415, 2004) offered two pain transduction mechanisms, one being TRPV1/2-independent and the other TRPV1-dependent. The former detects noxious heat under normal conditions without the presence of TRPV1 or TRPV2, and the latter requires TRPV1 under pathophysiological conditions. Unidentified isolectin B4 (IB4)-positive but TRPV1-negative small neurons with a higher noxious heat threshold are feasible, because a spliced isoform of TRPV1 responsive to noxious heat (47°C) but not responsive to either proton or capsaicin is present in human and rat sensory neurons. Thus, the IB4-positive but TRPV1-negative small sensory neurons must have a crucial role in the noxious heat response.

Comparison of the transport of QX-314 through TRPA1, TRPM8, and TRPV1 channels

Background It has been demonstrated that N-ethyl-lidocaine (QX-314) can target the transient receptor protein vanilloid 1 (TRPV1) nociceptors when coadministered with capsaicin, resulting in a selective block of the nociceptors. Capsaicin is problematic in therapeutic use because it induces firing of nociceptors. The present study aimed to search for substitutes for capsaicin. We also examined the transportability of QX-314 into nociceptive neurons, through the pores of transient receptor potential ankyrin 1 (TRPA1), transient receptor potential melastatin-8 (TRPM8), and TRPV1. Methods To investigate the effect on TRPA1, injections of a vehicle, allyl isothiocyanate (AITC), QX-314, or AITC/QX-314 were made into the hind paws of rats. The effects of menthol and capsaicin on the opening of TRPM8 and TRPV1 were also examined and compared with the potency of QX-314. To examine inhibition of the antinociceptive effect by capsaicin/ QX-314, capsazepine (50 μg/mL; 10 μL) was injected 30 minutes prior to capsaicin/QX-314 (10 μL) injection. Thermal sensitivity was investigated by the Hargreaves method. 5(6)-carboxyfluorescein (FAM)-conjugated QX-314 was used as a tracer to examine how many and which kind of dorsal root ganglia accumulate this molecule. QX-314-FAM, capsaicin/QX-314-FAM, AITC/QX-314-FAM, and menthol/QX-314-FAM were injected into the paw. Two weeks after injections, dorsal root ganglia were removed and sectioned with a cryostat. Results The capsaicin/QX-314 group induced longer withdrawal-response latency at 60 to 300 minutes after injection than the control. Both menthol only and menthol/QX-314 injections showed analgesia 10 to 60 minutes after injection. No significant difference was seen between the capsazepine/capsaicin/QX-314 group and the vehicle group. The fluorescence in small- and medium-sized neurons was conspicuous in only the dorsal root ganglia injected with capsaicin/ QX-314-FAM. Conclusion These results indicate that TRPA1 and TRPM8 are ineffective in the transport of QX-314 compared with TRPV1.

Electron microscopic study of the effect of capsaicin on the mouse chorda tympani nerves

Studies were made to determine whether this nerve contains capsaicin-sensitive fibres. Capsaicin (50 mg/kg, subcutaneously) was injected into 2 animals on day 2 after birth and into one animal on days 2 and 3 after birth. Both chorda tympani of these and 4 control mice were later excised. The constituent fibres of 3 of the capsaicin-treated and all 8 control nerves were then analysed. The myelinated fibres in 3 chorda tympani of treated and control animals were measured, and the unmyelinated axons in Schwann cells were counted from electron-micrograph montages of the entire nerve. Normal chorda tympani contained about 600 nerve fibres, 55% myelinated and 45% unmyelinated. Capsaicin-treatment did not change the constituent fibres nor the size distribution of the myelinated fibres. Thus no capsaicin-sensitive, nociceptive fibres were found in the mouse chorda tympani. Capsaicin does not destroy the neurones of the geniculate ganglion and parasympathetic, presynaptic fibres. Therefore, gustation and secretion of saliva are not influenced by capsaicin.

Quantitative electron-microscopic analyses of pulpal nerve fibres in the mouse lower incisor after neonatal capsaicin treatment

A single dose of capsaicin (50 mg/kg) was injected subcutaneously into four mice on day 2 of life; four untreated mice were used as controls. Six months later, a drop of 30 microM capsaicin was instilled on to the cornea of all the mice and the number of times the eyes were wiped was counted to assess the effect of capsaicin on trigeminal sensory neurones. Ultrathin cross-sections were made of the apical pulp of the incisors on both sides of control (n = 8) and capsaicin-treated animals (n = 8). Electron micrographs of pulp nerves were taken and enlarged to a final magnification of x34,000. The numbers of unmyelinated axons in the pulps of all 16 incisors and of unmyelinated axons per Schwann cell in the pulps of four incisors each from the control and capsaicin-treated groups were counted. The short diameters of unmyelinated axons were measured with a computer-operated image analyser. The number of eye wipings was eight-fold less in the capsaicin-treated than in the normal group. This finding clearly indicated that capsaicin irreversibly affected the chemogenic nociceptive trigeminal neurones. The mean number of unmyelinated axons was 345 in controls and 217 (37.1% reduction) in capsaicin-treated animals. The number of unmyelinated axons of less than 0.6 microns dia was 41.5% less in capsaicin-treated mice than in controls. Thus, fine unmyelinated axons in the mouse incisor pulp are capsaicin sensitive, and they are assumed to be nociceptive fibres conveying pain stimuli from the tooth. Capsaicin affected Schwann cells, even those with few unmyelinated axons.(ABSTRACT TRUNCATED AT 250 WORDS)

Corneal Nerve Fiber Structure, Its Role in Corneal Function, and Its Changes in Corneal Diseases

Recently, in vivo confocal microscopy is used to examine the human corneal nerve fibers morphology. Corneal nerve fiber architecture and its role are studied in healthy and pathological conditions. Corneal nerves of rats were studied by nonspecific acetylcholinesterase (NsAchE) staining. NsAchE-positive subepithelial (stromal) nerve fiber has been found to be insensitive to capsaicin. Besides, NsAchE-negative but capsaicin-sensitive subbasal nerve (leash) fibers formed thick mesh-like structure showing close interconnections and exhibit both isolectin B4- and transient receptor potential vanilloid channel 1- (TRPV1-) positive. TRPV1, TRPV3, TRPA (ankyrin) 1, and TRPM (melastatin) 8 are expressed in corneal nerve fibers. Besides the corneal nerve fibers, the expressions of TRPV (1, 3, and 4), TRPC (canonical) 4, and TRPM8 are demonstrated in the corneal epithelial cell membrane. The realization of the importance of TRP channels acting as polymodal sensors of environmental stresses has identified potential drug targets for corneal disease. The pathophysiological conditions of corneal diseases are associated with disruption of normal tissue innervation, especially capsaicin-sensitive small sensory nerve fibers. The relationships between subbasal corneal nerve fiber morphology and neurotrophic keratopathy in corneal diseases are well studied. The recommended treatment for neurotrophic keratopathy is administration of preservative free eye drops.

Roles of Glia, Immune Cells and the Thermo-TRP Channels, TRPV1, TRPA1 and TRPM8, in Pathological Pain

Studies into the interactions between glia/immune cells and neurons have focused on the induction of patho-logical (neuropathic or inflammatory) pain. Growing evidence of close relationships between peripheral and central glia and pathological pain has emerged during the last 2 decades. Numerous experimental studies have showed the release of cytokines and inflammatory neuropeptides from peripheral and central terminals of primary sensory neurons and from ac-tivated peripheral and central glia after nerve injury (crush, ligation or transection), which in turn act in a paracrine or autocrine manner. Cytokines induce the synthesis of algogens (pain-inducing substances such as prostaglandin) which leads to the primary (peripheral) or secondary (central) sensitization responsible for hyperalgesia or allodynia under in-flammatory conditions. The review has also highlighted the role of thermo transient receptor potential (TRP) channels TRPV1, TRPA1 and TRPM8 in the induction of pathological pain. The noxious heat sensor TRPV1 has an overt role in noxious heat hyperalgesia or allodynia, whereas TRPA1 and TRPM8 seem to have roles in noxious cold or mechanical al-lodynia, although results are inconsistent. Close mutual interrelationships between immune and glial cells and thermoTRP channels via cytokines or pro-inflammatory neuropeptides cannot be ignored when attempting to explain the induction and continuation of pathological pain. Investigations on the initial signals sent to the central area (superficial dorsal horn) remote from injured (or infectious) sites are a key point to clarify the mechanisms of pathological pain.