Megumi Funakubo

Reduction of TRPV1 expression in the trigeminal system by botulinum neurotoxin type-A.

Botulinum neurotoxin type-A (BoNT-A) is clinically used for patients with pain disorders and dystonia. The precise mechanism whereby BoNT-A controls pain remains elusive. Here, we studied how BoNT-A affects the expression of the transient receptor potential vanilloid subfamily member 1 (TRPV1), a cation channel critically implicated in nociception, in the trigeminal system. Histological studies revealed that subcutaneous BoNT-A injection (0.25, 0.5, or 5 ng/kg) into the face targeted the ophthalmic division of trigeminal ganglion (TG) neurons and decreased TRPV1-immunoreactive neurons in the TG and TRPV1-immunoreactive fibers in rat trigeminal terminals. Of note, TG neurons that received projections from the dura mater, a principal site of headache generation, had reduced TRPV1 expression. BoNT-A-induced cleavage of SNAP25 (synaptosomal-associated protein of 25-kDa) in the TG became obvious 2 days after BoNT-A administration and persisted for at least 14 days. Quantitative real-time RT-PCR (reverse transcription-polymerase chain reaction) data indicated that the TRPV1-decreasing effects of BoNT-A were not mediated by transcriptional downregulation. By employing a surface protein biotin-labeling assay, we demonstrated that BoNT-A inhibited TRPV1 trafficking to the plasma membrane in primary TG neurons. Moreover, Y200F-mutated TRPV1, which is incapable of trafficking to the plasma membrane, was expressed in PC12 cells by transfection, and pharmacological studies revealed that TRPV1 in the cytoplasm was more predisposed to proteasome-mediated proteolysis than plasma membrane-located TRPV1. We conclude that the mechanism by which BoNT-A reduces TRPV1 expression involves the inhibition of TRPV1 plasma membrane trafficking and proteasome-mediated degradation in the cytoplasm. This paradigm seems to explain how BoNT-A alleviates TRPV1-mediated pain. Our data reveal a likely molecular mechanism whereby BoNT-A treatment reduces TRPV1 expression in the trigeminal system and provide important clues to novel therapeutic measures for ameliorating craniofacial pain.

Transplantation of Bone Marrow-Derived Mononuclear Cells Improves Mechanical Hyperalgesia, Cold Allodynia and Nerve Function in Diabetic Neuropathy

Relief from painful diabetic neuropathy is an important clinical issue. We have previously shown that the transplantation of cultured endothelial progenitor cells or mesenchymal stem cells ameliorated diabetic neuropathy in rats. In this study, we investigated whether transplantation of freshly isolated bone marrow-derived mononuclear cells (BM-MNCs) alleviates neuropathic pain in the early stage of streptozotocin-induced diabetic rats. Two weeks after STZ injection, BM-MNCs or vehicle saline were injected into the unilateral hind limb muscles. Mechanical hyperalgesia and cold allodynia in SD rats were measured as the number of foot withdrawals to von Frey hair stimulation and acetone application, respectively. Two weeks after the BM-MNC transplantation, sciatic motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV), sciatic nerve blood flow (SNBF), mRNA expressions and histology were assessed. The BM-MNC transplantation significantly ameliorated mechanical hyperalgesia and cold allodynia in the BM-MNC-injected side. Furthermore, the slowed MNCV/SNCV and decreased SNBF in diabetic rats were improved in the BM-MNC-injected side. BM-MNC transplantation improved the decreased mRNA expression of NT-3 and number of microvessels in the hind limb muscles. There was no distinct effect of BM-MNC transplantation on the intraepidermal nerve fiber density. These results suggest that autologous transplantation of BM-MNCs could be a novel strategy for the treatment of painful diabetic neuropathy.

Changes in cardiovascular parameters and plasma norepinephrine level in rats after chronic constriction injury on the sciatic nerve

&NA; To evaluate whether neuropathic pain affects autonomic nervous activities, we investigated daily change in cardiovascular parameters and plasma norepinephrine (NE) in free‐moving rats after chronic constriction injury (CCI) on the sciatic nerve. Arterial blood pressure (BP), heart rate (HR), and the power spectrum of pulse interval variability were analyzed. Daily change in motor activity and nociceptive behavior was also measured from some CCI rats. In others, NE from daily blood samples was quantified and spontaneous pain was evaluated by daily monitoring of foot guarding behavior. We identified three stages in the daily change of cardiovascular parameters and plasma NE level over 3 weeks following CCI. The first stage (up to 3 days after the surgery) was characterized by increased MAP and HR, especially in the daytime, even though plasma NE was unchanged and motor activity decreased. The second stage (mid first to mid second postoperative weeks) was characterized by increased daytime MAP and HR, and the animals developed punctate hyperalgesia in the affected hindpaw. An NE surge that may have been related to spontaneous pain was present 3–5 days after CCI. The third stage, which appeared after the second postoperative week, was characterized by normalized MAP and decreased HR, and increased high‐frequency (0.8–3.0 Hz) power in pulse interval variability, which is an index of cardiac parasympathetic tone. These results demonstrated that cardiovascular function was kept high through sympathetic and non‐sympathetic activity for 2 weeks after CCI, followed by a predominance of parasympathetic tone.

Increases in Neuronal Activity in Rat Spinal Trigeminal Nucleus Following Changes in Barometric Pressure—Relevance for Weather-Associated Headaches?

Objective.— To determine whether controlled changes in barometric pressure activate rat spinal trigeminal neurons as a possible animal correlate of headaches.

Artificially produced meteorological changes aggravate pain in adjuvant-induced arthritic rats.

To examine the effects of change in meteorological parameters on pain-related behaviors in a simulated arthritic condition, rats with an injection of complete Freunds adjuvant into the tibio-tarsal joint were exposed to low barometric pressure (20 mmHg below the natural atmospheric pressure) and low ambient temperature (7 degrees C lower than 22 degrees C) in a climate-controlled room. When the arthritic rats were exposed to these environments, the already increased number of hindpaw withdrawals in response to noxious mechanical stimulation (hyperalgesia) was further increased, and a hindpaw withdrawal response to innocuous mechanical stimulation (allodynia) began to occur. Such exposures did not influence any of the pain-related behaviors of the control rats. These results show that lowering barometric pressure and ambient temperature within the range of natural environmental fluctuation intensify pain in arthritic rats.

Activation of extracellular signal-regulated kinase in the trigeminal ganglion following both treatment of the dura mater with capsaicin and cortical spreading depression

Extracellular signal-regulated kinase (ERK) is known to be phosphorylated after exposure to noxious stimuli. In this study, we investigated the response in the dura mater to nociceptive stimulation, which is thought to be responsible for the pathogenesis of headaches, including migraines. We also examined the level of ERK phosphorylation in the trigeminal ganglion following cortical spreading depression (CSD), which is thought to play an important role in migraine pathophysiology. Western blot and immunohistochemical analyses showed a significant increase in the ERK phosphorylation levels 3 min following an application of 10mM capsaicin to the dura mater. This increase was inhibited after an application of the TRPV1 antagonist capsazepine or a MEK inhibitor. An immunohistochemical analysis revealed that most of the small-sized trigeminal ganglion neurons with TRPV1-immunoreactivity that innervate the dura mater exhibited pERK-immunoreactivity, suggesting that these neurons had responded to nociceptive stimulation. CSD increased the level of ERK phosphorylation 30 min after its elicitation, and this response was inhibited by a prior intraventricular administration of TRPV1 antagonist. These results indicate that CSD can activate dural TRPV1 to send nociceptive signals to the trigeminal system, and they provide important clues regarding the relationship between CSD and the trigeminovascular system.

Lowering barometric pressure aggravates depression-like behavior in rats

Weather change has been known to influence the condition of patients with mood disorder. However, no animal studies have tested the influence of climatic factor on emotional impairment. In this study, we examined the effect of lowering barometric pressure (LP) in a climate-controlled room on immobility time in the forced swim test in rats, which is considered to be an index of behavioral despair (helplessness). When the rats were exposed to daily repeated forced swim, the immobility time gradually increased. This increment was inhibited by repeated administration of the antidepressant imipramine, suggesting that the immobility is an anxiety/depression-like behavior. LP exposure (20 hPa below the natural atmospheric pressure) further increased immobility time in rats submitted to repeated forced swim. In another series of experiments, we examined the effect of daily repeated LP exposure on the maintenance of immobility after withdrawal from 6-day repeated forced swim. When the rats were challenged with forced swim under natural atmospheric pressure on day 14 after the withdrawal, immobility time was significantly longer than in non-conditioned rats. These findings demonstrated that LP in the range of natural weather change augmented the depression-like behavior in rats.

The inner ear is involved in the aggravation of nociceptive behavior induced by lowering barometric pressure of nerve injured rats

Patients suffering from neuropathic pain often complain of pain aggravation when the weather is changing. The exact mechanism for weather change‐induced pain has not been clarified. We have previously demonstrated that experimentally lowering barometric pressure (LP) intensifies pain‐related behaviors in rats with chronic constriction injury (CCI). In the present experiment we examined whether this pain aggravating effect of LP exposure in nerve injured rats is still present after lesioning of the inner ear. We used both CCI and spinal nerve ligation (SNL) models for this study. We injected into the middle ear sodium arsanilate solution (100 mg/ml, 50 μl/ear), which is known to degenerate vestibular hair cells, under anesthesia the day before surgery. Rats were exposed to LP (27 hPa decrease over 8 min) 7–9 days after CCI or 5–8 days after SNL surgery, and pain‐related behavior (number of paw lifts induced by von Frey hair stimuli) was measured. When the inner ear lesioned SNL or CCI rats were exposed to LP, they showed no augmentation of pain‐related behavior. On the other hand, the pain aggravating effect of a temperature decrease (from 24 to 17 °C) was maintained in both SNL and CCI rats. These results suggest that the barometric sensor/sensing system influencing nociceptive behavior during LP in rats is located in the inner ear.

Alterations in microglia and astrocytes in the trigeminal nucleus caudalis by repetitive TRPV1 stimulation on the trigeminal nociceptors.

TRPV1 is a nonselective cation channel in nociceptors. TRPV1 stimulation has been shown to lead to the activation of microglia and astrocytes in the dorsal horn of the spinal cord. However, information on the effect of TRPV1 stimulation on glial activation in the trigeminal nucleus caudalis (TNC) is lacking. Here, we stimulated TRPV1 in the trigeminal afferents by a repetitive injection of 10 mmol/l capsaicin into the whisker pad for 2 days (d2 group), 4 days (d4 group), or 6 days (d6 group). As a control (c group), the vehicle was injected for 2 days. Anti-Iba1 and anti-glial fibrillary acidic protein antibodies were used to immunostain microglia and astrocytes in the TNC, respectively. The ratio of the cross-sectional area immunoreactive for Iba1 to the entire area of the TNC was increased in the d2 group compared with the c group on the injected side. Microglia were recruited to the superficial layers of the TNC. The numbers of microglia were reduced in the d4 group and the d6 group compared with the d2 group. The ratio of the cross-sectional area immunoreactive for glial fibrillary acidic protein to the entire TNC showed a significant increase in d2 group and the d4 group compared with the c group on the injected side. Behavioral analysis indicated that mechanical allodynia began to develop after 2 days of capsaicin treatment and persisted for at least 6 days after the onset of the repetitive capsaicin injection. These data indicate that TRPV1 stimulation activates the microglia and astrocytes in temporally distinct ways and that the development of mechanical allodynia is independent of such glial activation.

Low barometric pressure aggravates neuropathic pain in guinea pigs.

Several clinical studies have demonstrated a consistent relationship between changes in meteorological factors, particularly barometric pressure, and pain intensity in subjects with chronic pain. We have previously demonstrated that exposure to artificially low barometric pressure (LP) intensifies pain-related behaviors in rats with neuropathic pain. In the present study, guinea pigs with unilateral L5 spinal nerve ligation (SNL) were placed in a pressure-controlled chamber and subjected to LP of 10 or 27hPa below the ambient pressure. The SNL surgery led to increased hindpaw withdrawal frequencies to 34-, 59-, and 239-mN von Frey filaments (VFFs). When the SNL animals were subjected to both LP exposures consecutively, the hindpaw withdrawal frequencies further increased; the effect was most significant when the animals were exposed to LP 27hPa below ambient pressure. In contrast, no change was seen in a group of sham-operated control animals. These results indicate that fluctuations in LP within the range of natural weather patterns can potentiate neuropathic pain in guinea pigs.