Christopher M. Flores

Selective Blockade of the Capsaicin Receptor TRPV1 Attenuates Bone Cancer Pain

Cancer colonization of bone leads to the activation of osteoclasts, thereby producing local tissue acidosis and bone resorption. This process may contribute to the generation of both ongoing and movement-evoked pain, resulting from the activation of sensory neurons that detect noxious stimuli (nociceptors). The capsaicin receptor TRPV1 (transient receptor potential vanilloid subtype 1) is a cation channel expressed by nociceptors that detects multiple pain-producing stimuli, including noxious heat and extracellular protons, raising the possibility that it is an important mediator of bone cancer pain via its capacity to detect osteoclast- and tumor-mediated tissue acidosis. Here, we show that TRPV1 is present on sensory neuron fibers that innervate the mouse femur and that, in an in vivo model of bone cancer pain, acute or chronic administration of a TRPV1 antagonist or disruption of the TRPV1 gene results in a significant attenuation of both ongoing and movement-evoked nocifensive behaviors. Administration of the antagonist had similar efficacy in reducing early, moderate, and severe pain-related responses, suggesting that TRPV1 may be a novel target for pharmacological treatment of chronic pain states associated with bone cancer metastasis.

Epidemiological Evaluation of the Outcomes of Nonsurgical Root Canal Treatment in a Large Cohort of Insured Dental Patients

Selected outcomes following initial nonsurgical root canal treatment (NSRCT) procedures were retrospectively assessed using an insurance company database of 110,766 nonsurgical root canal procedures that were completed by endodontists and their referring general dentists. A subset of 44,613 cases, with a minimum required follow-up time of 2 yr, showed incidences of extraction, retreatment and periradicular surgery equal to 5.56%, 2.47%, and 1.41%, respectively. The incidence of subsequent extraction increased with patient age. Teeth that were not restored after root canal therapy were significantly more likely to undergo extraction than restored teeth. Although the practice pattern for endodontists consisted of a significantly higher proportion of molars (48% more; p < 0.001) and a smaller proportion of anterior teeth (43% less; p < 0.001) than general dentists, both groups of providers had comparable rates of untoward events. These data strongly support the hypothesis that the specialist practice provides similar rates of clinical success compared with other providers, even when treating significantly more complex NSRCT cases. Overall, 94.44% of nonsurgical root canal treated teeth remained functional over an average follow-up time of 3.5 yr. These results are an important indication of the benefits of endodontic treatment when provided in an integrated health care delivery system of endodontists and their referring general dentists.

Modulation of trigeminal sensory neuron activity by the dual cannabinoid–vanilloid agonists anandamide, N‐arachidonoyl‐dopamine and arachidonyl‐2‐chloroethylamide

Peripheral cannabinoids have been shown to suppress nociceptive neurotransmission in a number of behavioral and neurophysiological studies. It is not known, however, whether cannabinoids exert this action through direct interactions with nociceptors in the periphery and/or if other processes are involved. To gain a better understanding of the direct actions of cannabinoid‐vanilloid agonists on sensory neurons, we examined the effects of these compounds on trigeminal ganglion (TG) neurons in vitro. AEA (EC50=11.0 μM), NADA (EC50=857 nM) and arachidonyl‐2‐chloroethylamide ACEA (EC50=14.0 μM) each evoked calcitonin gene‐related peptide (CGRP) release from TG neurons. The TRPV1 antagonists iodo‐resiniferatoxin (I‐RTX) and capsazepine (CPZ) each obtunded AEA‐, NADA‐, ACEA‐ and capsaicin (CAP)‐evoked CGRP release with individually equivalent IC50s for each of the compounds (I‐RTX IC50 range=2.6–4.0 nM; CPZ IC50 range=523–1140 μM). The pro‐inflammatory mediator prostaglandin E2 significantly increased the maximal effect of AEA‐evoked CGRP release without altering the EC50. AEA, ACEA and CAP stimulated cAMP accumulation in TG neurons in a calcium‐ and TRPV1‐dependent fashion. Moreover, the protein kinase inhibitor staurosporine significantly inhibited AEA‐ and CAP‐evoked CGRP release. The pungency of AEA, NADA, ACEA and CAP in the rat eye‐wipe assay was also assessed. Interestingly, when applied intraocularly, NADA or CAP each produced nocifensive responses, while AEA or ACEA did not. Finally, the potential inhibitory effects of these cannabinoids on TG nociceptors were evaluated. Neither AEA nor ACEA decreased CAP‐evoked CGRP release. Furthermore, neither of the cannabinoid receptor type 1 antagonists SR141716A nor AM251 had any impact on either basal or CAP‐evoked CGRP release. AEA also did not inhibit 50 mM K+‐evoked CGRP release and did not influence bradykinin‐stimulated inositol phosphate accumulation. We conclude that the major action of AEA, NADA and ACEA on TG neurons is excitatory, while, of these, only NADA is pungent. These findings are discussed in relation to our current understanding of interactions between the cannabinoid and vanilloid systems and nociceptive processing in the periphery.

The neuronal distribution of cannabinoid receptor type 1 in the trigeminal ganglion of the rat

Cannabinoid compounds have been shown to produce antinociception and antihyperalgesia by acting upon cannabinoid receptors located in both the CNS and the periphery. A potential mechanism by which cannabinoids could inhibit nociception in the periphery is the activation of cannabinoid receptors located on one or more classes of primary nociceptive neurons. To address this hypothesis, we evaluated the neuronal distribution of cannabinoid receptor type 1 (CB1) in the trigeminal ganglion (TG) of the adult rat through combined in situ hybridization (ISH) and immunohistochemistry (IHC). CB1 receptor mRNA was localized mainly to medium and large diameter neurons of the maxillary and mandibular branches of the TG. Consistent with this distribution, in a de facto nociceptive sensory neuron population that exhibited vanilloid receptor type 1 immunoreactivity, colocalization with CB1 mRNA was also sparse (<5%). Furthermore, very few neurons (approximately 5%) in the peptidergic (defined as calcitonin gene-related peptide- or substance P-immunoreactive) or the isolectin B4-binding sensory neuron populations contained CB1 mRNA. In contrast, and consistent with the neuron-size distribution for CB1, nearly 75% of CB1-positive neurons exhibited N52-immunoreactivity, a marker of myelinated axons. These results indicate that in the rat TG, CB1 receptors are expressed predominantly in neurons that are not thought to subserve nociceptive neurotransmission in the noninjured animal. Taken together with the absence of an above background in situ signal for CB2 mRNA in TG neurons, these findings suggest that the peripherally mediated antinociceptive effects of cannabinoids may involve either as yet unidentified receptors or interaction with afferent neuron populations that normally subserve non-nociceptive functions.

Treatment of trigeminal ganglion neurons in vitro with NGF, GDNF or BDNF: effects on neuronal survival, neurochemical properties and TRPV1-mediated neuropeptide secretion

BackgroundNerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) all play important roles in the development of the peripheral sensory nervous system. Additionally, these growth factors are proposed to modulate the properties of the sensory system in the adult under pathological conditions brought about by nerve injury or inflammation. We have examined the effects of NGF, GDNF and BDNF on adult rat trigeminal ganglion (TG) neurons in culture to gain a better understanding of how these growth factors alter the cytochemical and functional phenotype of these neurons, with special attention to properties associated with nociception.ResultsCompared with no growth factor controls, GDNF, at 1 and 100 ng/ml, significantly increased by nearly 100% the number of neurons in culture at 5 days post-plating. A significant, positive, linear trend of increasing neuron number as a function of BDNF concentration was observed, also peaking at nearly 100%. NGF treatment was without effect. Chronic treatment with NGF and GDNF significantly and concentration-dependently increased 100 nM capsaicin (CAP)-evoked calcitonin gene-related peptide (CGRP) release, reaching approximately 300% at the highest concentration tested (100 ng/ml). Also, NGF and GDNF each augmented anandamide (AEA)- and arachidonyl-2-chloroethylamide (ACEA)-evoked CGRP release, while BDNF was without effect. Utilizing immunohistochemistry to account for the proportions of TRPV1- or CGRP-positive neurons under each growth factor treatment condition and then standardizing evoked CGRP release to these proportions, we observed that NGF was much more effective in enhancing CAP- and 50 mM K+-evoked CGRP release than was GDNF. Furthermore, NGF and GDNF each altered the concentration-response function for CAP- and AEA-evoked CGRP release, increasing the Emax without altering the EC50 for either compound.ConclusionsTaken together, our results illustrate that NGF, GDNF and BDNF differentially alter TG sensory neuron survival, neurochemical properties and TRPV1-mediated neuropeptide release in culture. In particular, our findings suggest that GDNF and NGF differentially modulate TRPV1-mediated neuropeptide secretion sensitivity, with NGF having a much greater effect on a per neuron basis than GDNF. These findings are discussed in relation to possible therapeutic roles for growth factors or their modulators in pathological pain states, especially as these relate to the trigeminal system.

An In Vitro Evaluation of the Accuracy of the Root ZX in the Presence of Various Irrigants

The purpose of this study was to evaluate the accuracy of the Root ZX in vitro in the presence of a variety of endodontic irrigants. The in vitro model, described by Donnelly, consisted of refrigerated gelatin made with 0.9% sodium chloride instead of water. The following irrigants were tested: 2% lidocaine with 1:100,000 epinephrine, 5.25% sodium hypochlorite, RC Prep, liquid EDTA, 3% hydrogen peroxide, and Peridex. A total of 30 extracted, single-rooted teeth were used. The experimental measurements in the presence of the various irrigants were compared with the actual canal lengths. The present data indicate that the Root ZX electronic apex locator reliably measured canal lengths to within 0.31 mm and that there was virtually no difference in the length determination as a function of the seven irrigants used. These results strongly support the concept that the Root ZX is a useful, versatile, and accurate device for the determination of canal lengths over a wide range of irrigants commonly used in the practice of endodontics.

Capsaicin-evoked release of immunoreactive calcitonin gene-related peptide from rat trigeminal ganglion: evidence for intraganglionic neurotransmission.

&NA; Chemically‐mediated cross‐excitation has been described between neurons within sensory ganglia. However, the identity and source of the chemical mediators is not known. Ca2+‐dependent release of neurotransmitters from cultured sensory neurons in vitro has been observed, although neurite outgrowth may confound the ability to extrapolate findings from culture systems to in vivo conditions. Thus, the present studies evaluate the hypothesis of capsaicin‐sensitive intraganglionic neuropeptide release from freshly prepared slices of rat sensory ganglia. The ganglionic slice preparation provides an advantage over neuronal cultures, because release may be assessed within minutes after tissue collection (minimizing phenotypic changes) and while maintaining gross anatomical relationships. Trigeminal ganglia (TGG) were quickly removed from male, Sprague–Dawley rats (175–200 g), chopped into 200 &mgr;m slices and placed into chambers within 3 min of collection. Chambers were perfused with buffer, and superfusates were collected and assayed for immunoreactive calcitonin gene‐related peptide (iCGRP) release via radioimmunoassay. After about 90 min of baseline collection, tissue was treated with capsaicin followed by a washout period. Capsaicin (1–100 &mgr;M) evoked concentration‐dependent increases in iCGRP release. A competitive capsaicin receptor antagonist, capsazepine, significantly inhibited capsaicin‐evoked release of iCGRP. In addition, capsaicin‐evoked release of iCGRP was dependent on the presence of extracellular calcium. Furthermore, capsaicin‐evoked release from TGG slices was significantly greater than that from slices of equivalent weights of adjacent trigeminal nerve shown histologically to be free of neuronal somata. These data support the hypothesis that Ca2+‐dependent exocytosis of neuropeptides may occur within the TGG in vivo and that the majority of this release derives from neuronal somata.

Cholinergic modulation of nociceptive responses in vivo and neuropeptide release in vitro at the level of the primary sensory neuron

&NA; Muscarinic acetylcholine receptors (mAChRs) have been widely reported as pharmacological targets for the treatment of pain. However, most of these efforts have focused on CNS mAChRs and their role in modulating nociception at the level of the spinal cord. The present study examines the contribution of peripheral mAChRs in trigeminal nociceptive pathways using a combination of in vivo and in vitro approaches. In the formalin model of orofacial nociception in rats, a peri‐oral co‐injection of the M2 agonist arecaidine dose‐dependently inhibited phase 2 nocifensive behavior up to approximately 50% at 5 nmol. This effect was blocked by co‐treatment with the mAChR antagonist atropine and was not seen when arecaidine was administered under the skin of the back, a site distant from that of the formalin injection. In vitro superfusion of isolated rat buccal mucosa with the non‐selective mAChR agonist muscarine or arecaidine led to a concentration‐dependent inhibition of capsaicin‐evoked CGRP release to 39% (EC50=255 nM) and 28% (EC50=847 nM) of control values, respectively. Both responses were blocked by the non‐selective mAChR antagonist atropine or the M2 antagonist gallamine. Further, the endogenous ligand ACh produced a bi‐phasic response, potentiating evoked CGRP release to 195% of control (EC50=918 nM) and inhibiting evoked CGRP release to 45% of control (EC50=255 &mgr;M), effects that were shown to be mediated by nAChRs and mAChRs, respectively. Finally, combined in situ hybridization/immunofluorescence demonstrated that m2 mRNA was present in 20% of trigeminal ganglion neurons between 30 and 60 &mgr;m in diameter and that 5–9% of these also expressed CGRP or VR1 immunoreactivity. These results show that activation of peripheral M2 receptors produces antinociception in vivo and the inhibition of nociceptor activity in vitro. While histological analyses at the level of the trigeminal neuronal cell bodies leave open the question of whether the effects of M2 agonists are direct or indirect, these data indicate that primary sensory neuronal M2 receptors may represent a viable peripheral target for the treatment of pain and inflammation.

Targeted disruption of the voltage-dependent calcium channel α2/δ-1-subunit

Cardiac L-type voltage-dependent Ca(2+) channels are heteromultimeric polypeptide complexes of alpha(1)-, alpha(2)/delta-, and beta-subunits. The alpha(2)/delta-1-subunit possesses a stereoselective, high-affinity binding site for gabapentin, widely used to treat epilepsy and postherpetic neuralgic pain as well as sleep disorders. Mutations in alpha(2)/delta-subunits of voltage-dependent Ca(2+) channels have been associated with different diseases, including epilepsy. Multiple heterologous coexpression systems have been used to study the effects of the deletion of the alpha(2)/delta-1-subunit, but attempts at a conventional knockout animal model have been ineffective. We report the development of a viable conventional knockout mouse using a construct targeting exon 2 of alpha(2)/delta-1. While the deletion of the subunit is not lethal, these animals lack high-affinity gabapentin binding sites and demonstrate a significantly decreased basal myocardial contractility and relaxation and a decreased L-type Ca(2+) current peak current amplitude. This is a novel model for studying the function of the alpha(2)/delta-1-subunit and will be of importance in the development of new pharmacological therapies.

The RNA binding and transport proteins staufen and fragile X mental retardation protein are expressed by rat primary afferent neurons and localize to peripheral and central axons.

Neuronal proteins have been traditionally viewed as being derived solely from the soma; however, accumulating evidence indicates that dendritic and axonal sites are capable of a more autonomous role in terms of new protein synthesis. Such extra-somal translation allows for more rapid, on-demand regulation of neuronal structure and function than would otherwise be possible. While mechanisms of dendritic RNA transport have been elucidated, it remains unclear how RNA is trafficked into the axon for this purpose. Primary afferent neurons of the dorsal root (DRG) and trigeminal (TG) ganglia have among the longest axons in the neuraxis and such axonal protein synthesis would be advantageous, given the greater time involved for protein trafficking to occur via axonal transport. Therefore, we hypothesized that these primary sensory neurons might express proteins involved in RNA transport. Rat DRG and TG neurons expressed staufen (stau) 1 and 2 (detected at the mRNA level) and stau2 and fragile x mental retardation protein (FMRP; detected at the protein level). Stau2 mRNA was also detected in human TG neurons. Stau2 and FMRP protein were localized to the sciatic nerve and dorsal roots by immunohistochemistry and to dorsal roots by Western blot. Stau2 and FMRP immunoreactivities colocalized with transient receptor potential channel type 1 immunoreactivity in sensory axons of the sciatic nerve and dorsal root, suggesting that these proteins are being transported into the peripheral and central terminals of nociceptive sensory axons. Based on these findings, we propose that stau2 and FMRP proteins are attractive candidates to subserve RNA transport in sensory neurons, linking somal transcriptional events to axonal translation.