Andrew J. Mannes

Deletion of vanilloid receptor 1-expressing primary afferent neurons for pain control

Control of cancer, neuropathic, and postoperative pain is frequently inadequate or compromised by debilitating side effects. Inhibition or removal of certain nociceptive neurons, while retaining all other sensory modalities and motor function, would represent a new therapeutic approach to control severe pain. The enriched expression of transient receptor potential cation channel, subfamily V, member 1 (TRPV1; also known as the vanilloid receptor, VR1) in nociceptive neurons of the dorsal root and trigeminal ganglia allowed us to test this concept. Administration of the potent TRPV1 agonist resiniferatoxin (RTX) to neuronal perikarya induces calcium cytotoxicity by opening the TRPV1 ion channel and selectively ablates nociceptive neurons. This treatment blocks experimental inflammatory hyperalgesia and neurogenic inflammation in rats and naturally occurring cancer and debilitating arthritic pain in dogs. Sensations of touch, proprioception, and high-threshold mechanosensitive nociception, as well as locomotor function, remained intact in both species. In separate experiments directed at postoperative pain control, subcutaneous administration of RTX transiently disrupted nociceptive nerve endings, yielding reversible analgesia. In human dorsal root ganglion cultures, RTX induced a prolonged increase in intracellular calcium in vanilloid-sensitive neurons, while leaving other, adjacent neurons unaffected. The results suggest that nociceptive neuronal or nerve terminal deletion will be effective and broadly applicable as strategies for pain management.

Physiologic and antinociceptive effects of intrathecal resiniferatoxin in a canine bone cancer model

Background:Resiniferatoxin is a potent capsaicin analog. Intrathecal administration leads to selective, prolonged opening of the transient receptor potential V1 ion channel, which is localized mainly to C-fiber primary afferent nociceptive sensory neurons. Following work in laboratory animals, the authors explored the use of intrathecal resiniferatoxin to control spontaneous bone cancer pain in companion (pet) dogs. Methods:Normal canine population: Behavioral testing was performed to establish baseline paw withdrawal latency; subsequently, general anesthesia was induced and resiniferatoxin was administered intrathecally while hemodynamic parameters were recorded. Behavior testing was repeated for 12 days after administration of resiniferatoxin. Clinical canine population: Twenty companion dogs with bone cancer pain were recruited. The animal’s baseline level of discomfort and analgesic use were recorded. Resiniferatoxin was administered intrathecally and hemodynamic parameters were monitored while the dogs were under general anesthesia. Dogs were reevaluated up to 14 weeks after resiniferatoxin administration. Results:Normal canine population: In the first minutes after resiniferatoxin injection, there were significant (P < 0.05) increases in mean arterial blood pressure and heart rate from baseline. Two days after injection, limb withdrawal latencies increased to the point of cutoff in the dogs that received at least 1.2 &mgr;g/kg resiniferatoxin. Clinical canine population: From baseline, there were significant (P < 0.05) increases in mean arterial blood pressure and heart rate after resiniferatoxin injection. Comfort scores were significantly improved at 2, 6, 10, and 14 weeks after resiniferatoxin administration (P < 0.0001). There was decreased or discontinued use of supplemental analgesics in 67% of the dogs 2 weeks after resiniferatoxin administration. Conclusions:Intrathecal resiniferatoxin elicits transient hemodynamic effects. In controls, a profound and sustained blockade of thermal stimuli is produced in a dose-dependent fashion. Similar administration in dogs with bone cancer produces a prolonged antinociceptive response.

A Paracrine Paradigm for in Vivo Gene Therapy in the Central Nervous System: Treatment of Chronic Pain

A limitation of current gene therapy efforts aimed at central nervous system disorders concerns distribution of vectors on direct injection into neural tissue. Here we have circumvented this problem by transferring genes to the meninges surrounding the spinal cord, achieving an in vivo gene transfer paradigm for treating chronic pain. The therapeutic vector consisted of a recombinant adenovirus encoding a secreted form of the potent endogenous opioid beta-endorphin. In an inflammation model of persistent pain, administration of the vector into the cerebrospinal fluid (CSF) surrounding the spinal cord transduced meningeal pia mater cells. The resulting increase in beta-endorphin secretion attenuated inflammatory hyperalgesia, yet had no effect on basal nociceptive responses. This demonstration of a gene transfer approach to pain treatment can be generalized to neurodegenerative disorders in which broad spatial distribution of therapeutic effect is critical.

Actions of intrathecal diphtheria toxin-substance P fusion protein on models of persistent pain.

Substance P (SP) plays a central role in the transduction of second messenger signals from primary afferent nociceptive terminals to second-order neurons in the spinal cord. We have tested a recombinant engineered diphtheria toxin/SP fusion protein (DAB389SP) in acute and chronic pain models in the rat. DAB389SP binds to the SP receptor (SPR) and is internalized and kills SPR-expressing cells by blocking cellular protein synthesis. DAB389SP delivery was by intrathecal infusion, of varying duration, at the lumbar level. In the chronic constriction injury model of neuropathic pain a significant reduction in mechanically induced hyperalgesia was obtained. This effect was less marked in an acute carageenan inflammation model. Although other pain characteristics (mechano-allodynia, cold-allodynia, and heat-hyperalgesia) showed some improvement, these were less pronounced. Immunocytochemistry revealed a toxin-induced reduction in lamina I, of SPR and of NMDA NR1 subunit receptor expressing neurons, and of c-Fos, an inducible molecular marker of persistent nociceptive activity. The use of cytotoxic fusion proteins to target specific cell types may be of considerable benefit in the study of nociception and the treatment of chronic pain.

The Vanilloid Agonist Resiniferatoxin for Interventional-Based Pain Control

The idea of selectively targeting nociceptive transmission at the level of the peripheral nervous system is attractive from multiple perspectives, particularly the potential lack of non-specific (non-targeted) CNS side effects. Out of the multiple TRP channels involved in nociception, TRPV1 is a strong candidate based on its biophysical conductance properties and its expression in inflammation-sensitive dorsal root ganglion neurons and their axons and central and peripheral nerve terminals. While TRPV1 antagonists have undergone extensive medicinal chemical and pharmacological investigation, for TRPV1 agonists nature has provided an optimized compound in RTX. RTX is not suitable for systemic administration, but it is highly adaptable to a variety of pain problems when used by local administration. This can include routes as diverse as subcutaneous, intraganglionic or intrathecal (CSF space around the spinal cord). The present review focuses on the molecular and preclinical animal experiments that form the underpinnings of our clinical trial of intrathecal RTX for pain in advanced cancer. As such this represents a new approach to pain control that emerges from a long line of research on capsaicin and other vanilloids, their physiological actions, and the molecular biology of the capsaicin receptor TRPV1.

Cystatin C as a cerebrospinal fluid biomarker for pain in humans.

&NA; Through a process of subtraction cloning and differential hybridization, we previously identified several new genes whose expression was induced by peripheral inflammation. One of these coded for cystatin C, a secreted cysteine protease inhibitor in the cystatin superfamily. We hypothesized that, concurrent with increased expression in dorsal horn, increased secretion would elevate the cystatin C content in cerebrospinal fluid (CSF) during active pain states. Alterations were assessed by immunoassay and by surface enhanced laser desorption ionization (SELDI) mass spectrometry with either reverse phase or immobilized anti‐cystatin C antibody surfaces using CSF from ten age‐matched obstetrical patients at term. Five control subjects were scheduled for an elective caesarian section and were not in pain. Another five subjects were in labor for 8.9±1 h and were in severe pain as assessed with a visual analog scale and the McGill short form questionnaire. The level of cystatin C as measured by immunoassay in the non‐pain patients was 2.77±0.75 &mgr;g/ml and in the pain patients 5.36±0.92 &mgr;g/ml (P<0.02). The elevation occurred without significant change in total CSF protein or &bgr;‐endorphin content. The cystatin C increase also was detectable by SELDI with either raw CSF or after antibody capture. These data are consistent with our previous animal study and the idea that persistent pain induces the synthesis and release of cystatin C in dorsal spinal cord, the surplus of which overflows into the CSF.

Molecular Signatures of Mouse TRPV1-Lineage Neurons Revealed by RNA-Seq Transcriptome Analysis

UNLABELLED Disorders of pain neural systems are frequently chronic and, when recalcitrant to treatment, can severely degrade the quality of life. The pain pathway begins with sensory neurons in dorsal root or trigeminal ganglia, and the neuronal subpopulations that express the transient receptor potential cation channel, subfamily V, member 1 (TRPV1) ion channel transduce sensations of painful heat and inflammation and play a fundamental role in clinical pain arising from cancer and arthritis. In the present study, we elucidate the complete transcriptomes of neurons from the TRPV1 lineage and a non-TRPV1 neuroglial population in sensory ganglia through the combined application of next-gen deep RNA-Seq, genetic neuronal labeling with fluorescence-activated cell sorting, or neuron-selective chemoablation. RNA-Seq accurately quantitates gene expression, a difficult parameter to determine with most other methods, especially for very low and very high expressed genes. Differentially expressed genes are present at every level of cellular function from the nucleus to the plasma membrane. We identified many ligand receptor pairs in the TRPV1 population, suggesting that autonomous presynaptic regulation may be a major regulatory mechanism in nociceptive neurons. The data define, in a quantitative, cell population-specific fashion, the molecular signature of a distinct and clinically important group of pain-sensing neurons and provide an overall framework for understanding the transcriptome of TRPV1 nociceptive neurons. PERSPECTIVE Next-gen RNA-Seq, combined with molecular genetics, provides a comprehensive and quantitative measurement of transcripts in TRPV1 lineage neurons and a contrasting transcriptome from non-TRPV1 neurons and cells. The transcriptome highlights previously unrecognized protein families, identifies multiple molecular circuits for excitatory or inhibitory autocrine and paracrine signaling, and suggests new combinatorial approaches to pain control.

Prolonged analgesic response of cornea to topical resiniferatoxin, a potent TRPV1 agonist

&NA; Analgesics currently available for the treatment of pain following ophthalmic surgery or injury are limited by transient effectiveness and undesirable or adverse side effects. The cornea is primarily innervated by small‐diameter C‐fiber sensory neurons expressing TRPV1 (transient receptor potential channel, subfamily V, member 1), a sodium/calcium cation channel expressed abundantly by nociceptive neurons and consequently a target for pain control. Resiniferatoxin (RTX), a potent TRPV1 agonist, produces transient analgesia when injected peripherally by inactivating TRPV1‐expressing nerve terminals through excessive calcium influx. The aim of the present study was to evaluate topical RTX as a corneal analgesic. In rat cornea, a single application of RTX dose dependently eliminated or reduced the capsaicin eye wipe response for 3–5 days, with normal nociceptive responses returning by 5–7 days. RTX alone produced a brief but intense noxious response, similar to capsaicin, necessitating pretreatment of the cornea with a local anesthetic. Topical lidocaine, applied prior to RTX, blocks acute nociceptive responses to RTX without impairing the subsequent analgesic effect. Importantly, RTX analgesia (a) did not impair epithelial wound healing, (b) left the blink reflex intact and (c) occurred without detectable histological damage to the cornea. Immunohistochemistry showed that loss of CGRP immunoreactivity, a surrogate marker for TRPV1‐expressing fibers, extended at least to the corneal‐scleral boundary and displayed a progressive return, coincident with the return of capsaicin sensitivity. These data suggest that RTX may be a safe and effective treatment for post‐operative or post‐injury ophthalmic pain.

Perineural resiniferatoxin selectively inhibits inflammatory hyperalgesia

Resiniferatoxin (RTX) is an ultrapotent capsaicin analog that binds to the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1). There is a large body of evidence supporting a role for TRPV1 in noxious-mediated and inflammatory hyperalgesic responses. In this study, we evaluated low, graded, doses of perineural RTX as a method for regional pain control. We hypothesized that this approach can provide long-term, but reversible, blockade of a portion of nociceptive afferent fibers within peripheral nerves when given at a site remote from the neuronal perikarya in the dorsal root ganglia. Following perineural RTX application to the sciatic nerve, we demonstrated a significant inhibition of inflammatory nociception that was dose- and time-dependent. At the same time, treated animals maintained normal proprioceptive sensations and motor control, and other nociceptive responses were largely unaffected. Using a range of mechanical and thermal algesic tests, we found that the most sensitive measure following perineural RTX administration was inhibition of inflammatory hyperalgesia. Recovery studies showed that physiologic sensory function could return as early as two weeks post-RTX treatment, however, immunohistochemical examination of the DRG revealed a partial, but significant reduction in the number of the TRPV1-positive neurons. We propose that this method could represent a beneficial treatment for a range of chronic pain problems, including neuropathic and inflammatory pain not responding to other therapies.

Quality of life and pain in premenopausal women with major depressive disorder: The POWER Study

BackgroundWhereas it is established that organic pain may induce depression, it is unclear whether pain is more common in healthy subjects with depression. We assessed the prevalence of pain in premenopausal women with major depression (MDD). Subjects were 21- to 45-year-old premenopausal women with MDD (N = 70; age: 35.4 +/- 6.6; mean +/- SD) and healthy matched controls (N = 36; age 35.4 +/- 6.4) participating in a study of bone turnover, the P.O.W.E.R. (P remenopausal, O steopenia/Osteoporosis, W omen, Ale ndronate, Depr ession) Study.MethodsPatients received a clinical assessment by a pain specialist, which included the administration of two standardized forms for pain, the Brief Pain Inventory – Short Form, and the Initial Pain Assessment Tool, and two scales of everyday stressors, the Hassles and Uplifts Scales. In addition, a quality-of-life instrument, the SF-36, was used. The diagnosis of MDD was established by a semi-structured interview, according to the DSM-IV criteria. Substance P (SP) and calcitonin-gene-related-peptide (CGRP), neuropeptides which are known mediators of pain, were measured every hour for 24 h in a subgroup of patients (N = 17) and controls (N = 14).ResultsApproximately one-half of the women with depression reported pain of mild intensity. Pain intensity was significantly correlated with the severity of depression (r2 = 0.076; P = 0.04) and tended to be correlated with the severity of anxiety, (r2 = 0.065; P = 0.07), and the number of depressive episodes (r2 = 0.072; P = 0.09). Women with MDD complained of fatigue, insomnia, and memory problems and experienced everyday negative stressors more frequently than controls. Quality of life was decreased in women with depression, as indicated by lower scores in the emotional and social well-being domains of the SF-36. SP (P < 0.0003) and CGRP (P < 0.0001) were higher in depressed subjects.ConclusionWomen with depression experienced pain more frequently than controls, had a lower quality of life, and complained more of daily stressors. Assessment of pain may be important in the clinical evaluation of women with MDD. SP and CGRP may be useful biological markers in women with MDD.