New technologies for studying the complexity of oral diseases.

Several new technologies are providing useful diagnostic tools and new information related to the pathogenesis of certain oral diseases. In this review, we describe several of these technologies including gene and microRNA arrays, proteomics, and antigen arrays as they relate to the study of Sjögren's syndrome and head and neck cancer. A common theme is the systematic analysis of large-scale inventories of RNAs, proteins, and autoantibody biomarkers revealing information not previously recognized. We also discuss metagenomic approaches that characterize the many different microorganisms present in the oral cavity that may impact oral and human health. Lastly, we describe applications of a new type of antibody-profiling technology termed Luciferase Immunoprecipitation Systems (LIPS), which has a wide dynamic range of detection of both linear and conformational epitopes needed for optimum diagnostics and biomarker discovery. We propose that the information offered by these technologies will enhance our ability to diagnose, treat, and further understand the pathogenesis of multiple oral diseases.

Salivary anti-Ro60 and anti-Ro52 antibody profiles to diagnose Sjogren's Syndrome.

Simple and non-invasive saliva-based diagnostics may be useful for the identification, understanding, and monitoring of autoimmune and infectious diseases. Previously, Luciferase Immunoprecipitation Systems (LIPS) were used for sensitive detection of patient serum autoantibodies in Sjögren's Syndrome (SjS), a chronic autoimmune disease affecting the salivary and lacrimal glands. Here we explored the ability of LIPS to diagnose SjS based on IgG autoantibodies in patient saliva. From LIPS testing, anti-Ro60 autoantibodies were detected in the saliva of 70% (19/27) of SjS patients with 96% specificity. Positive anti-Ro60 autoantibodies were also found in 70% of the matched serum samples (96% specificity). LIPS detected Ro52 autoantibodies in the saliva and serum of 67% of SjS patients with 100% specificity. Overall, the autoantibody titers in saliva were approximately 4000-fold lower by volume than serum, but still distinguished seropositive patients from controls. These results suggest that LIPS salivary-based testing for SjS autoantibodies is a practical alternative to serum and compatible with point-of-care testing.

Monocyte chemoattractant protein-1 in the choroid plexus: a potential link between vascular pro-inflammatory mediators and the CNS during peripheral tissue inflammation.

During peripheral tissue inflammation, inflammatory processes in the CNS can be initiated by blood-borne pro-inflammatory mediators. The choroid plexus, the site of cerebrospinal fluid (CSF) production, is a highly specialized interface between the vascular system and CNS, and thus, this structure may be an important element in communication between the vascular compartment and the CNS during peripheral tissue inflammation. We investigated the potential participation of the choroid plexus in this process during peripheral tissue inflammation by examining expression of the small inducible cytokine A2 (SCYA2) gene which codes for monocyte chemoattractant protein-1 (MCP-1). MCP-1 protein was previously reported to be induced in a variety of cells during peripheral tissue inflammation. In the basal state, SCYA2 is highly expressed in the choroid plexus as compared with other rat CNS tissues. During hind paw inflammation, SCYA2 expression was significantly elevated in choroid plexus, whereas it remained unchanged in a variety of brain regions. The SCYA2-expressing cells were strongly associated with the choroid plexus as vascular depletion of blood cells by whole-body saline flush did not significantly alter SCYA2 expression in the choroid plexus. In situ hybridization suggested that the SCYA2-expressing cells were localized to the choroid plexus stroma. To elucidate potential molecular mechanisms of SCYA2 increase, we examined genes in the nuclear factor-kappa B (NF-kappaB) signaling cascade including tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and inhibitor of kappa B alpha (IkappaBalpha) in choroid tissue. Given that we also detected increased levels of MCP-1 protein by ELISA, we sought to identify potential downstream targets of MCP-1 and observed altered expression levels of mRNAs encoding tight junction proteins TJP2 and claudin 5. Finally, we detected a substantial up-regulation of the transcript encoding endothelial leukocyte adhesion molecule 1 (E-selectin), a molecule which could participate in leukocyte recruitment to the choroid plexus along with MCP-1. Together, these results suggest that profound changes occur in the choroid plexus during peripheral tissue inflammation, likely initiated by blood-borne inflammatory mediators, which may modify events in CNS.

Genetic predictors for acute experimental cold and heat pain sensitivity in humans.

BACKGROUND: The genetic contribution to pain sensitivity underlies a complex composite of parallel pain pathways, multiple mechanisms, and diverse inter-individual pain experiences and expectations. METHODS: Variations for genes encoding receptors related to cold and heat sensation, such as transient receptor potential A subtype 1 (TRPA1), M subtype 8 (TRPM8), V subtype 1 (TRPV1), delta opioid receptor subtype 1 (OPRD1), catechol O-methyltransferase (COMT), and fatty acid amide hydrolyase (FAAH), were investigated in four major ethnic populations. RESULTS: We defined 13 haplotype blocks in European Americans, seven blocks in African Americans, seven blocks in Hispanic subjects, and 11 blocks in Asian Americans. Further study in European American subjects found significant associations between short duration cold pain sensitivity and variations in TRPA1, COMT, and FAAH in a gender dependent manner. Our observations demonstrate that genetic variations in TRPA1, COMT, and FAAH contribute gender specifically to individual variations in short duration cold pain sensitivity in a European American cohort. CONCLUSIONS: The effects of TRPA1 variations on experimental short duration heat pain sensitivity may contribute to inter-individual variation in pain sensitivity in humans.

Activation of spinal neuropeptide FF and the neuropeptide FF receptor 2 during inflammatory hyperalgesia in rats.

Several lines of evidence suggest that neuropeptide FF (NPFF) is involved in nociception and in the modulation of opioid-mediated analgesia. Following the identification of the precursor protein for NPFF, two NPFF receptors and a second PQRF-NH(2) containing peptide, termed NPVF, were identified. To further explore the functional role of PQRF-NH(2) peptides, we have studied their distribution and also the regulation of NPFF and NPVF systems in the spinal cord of rats with peripheral inflammation. The distribution of NPFF gene expression is very similar to that of NPFF immunoreactive peptide but is distinct from NPVF gene expression. In the rat spinal cord, gene expression of NPFF but not that of NPVF was up-regulated by persistent pain induced by carrageenan inflammation. The distribution of NPFF receptor 2 gene expression is very similar to that of the NPFF peptide with a striking localization in the superficial layer of spinal cord. In rats with carrageenan inflammation of the hind paw, expression of both NPFF and NPFF receptor 2 genes was up-regulated in the spinal cord, while expression of NPVF and NPFF receptor 1 genes was not affected. The results of this study demonstrate a coordinated involvement of the spinal NPFF system in the persistent nociceptive pain states. Several studies have found a potentiation and prolongation of morphine analgesia by NPFF, therefore, it is highly possible that the endogenous spinal NPFF system contributes to the enhanced analgesic potency of morphine in animals with peripheral inflammation.

A kinetic analysis of substance P trafficking.

The potential for administering substance P (SP) nocitoxins for the treatment of chronic pain has been identified. To characterize treatment protocols for the spinal cord or elsewhere, binding/internalization of these compounds at the cellular targets must be understood quantitatively. Thus, a kinetic model of SP binding and intracellular trafficking has been developed from data. The eight differential equation model describes surface binding between SP and neurokinin 1 receptor, clathrin-mediated endocytosis followed by spatial translation to a perinuclear endosome where SP is sorted from its receptor, SP degradation in late endosomes/early lysosomes, and return of sorted receptor to plasma membrane via recycling endosomes. With suitably optimized parameters, the model accounts for the kinetics of total, membrane-associated, and internalized SP in cells continuously exposed to SP, as well as the fractions of internalized SP remaining intact at 30 and 60 min. Simultaneously, the model accounts for the kinetics of internalization and receptor recycling after SP preloading of membrane and subsequent exposure to SP-free media. Rate constants (min(-1)) are: 0.034 +/- 0.004 (receptor off-rate), 0.15 +/- 0.03 (internalization), 0.048 +/- 0.003 (exit from sorting endosome), 0.062 +/- 0.008 (exit of labeled SP amino acids from prelysosome), and 0.029 +/- 0.004 (receptor return from recycling endosome to plasma membrane). The SP kinetics resemble those of transferrin and its receptor at the internalization step, but are several-fold slower in the sorting and recycling steps.

Hemispheric lateralization of somatosensory processing.

Processing of both painful and nonpainful somatosensory information is generally thought to be subserved by brain regions predominantly contralateral to the stimulated body region. However, lesions to right, but not left, posterior parietal cortex have been reported to produce a unilateral tactile neglect syndrome, suggesting that components of somatosensory information are preferentially processed in the right half of the brain. To better characterize right hemispheric lateralization of somatosensory processing, H(2)(15)O positron emission tomography (PET) of cerebral blood flow was used to map brain activation produced by contact thermal stimulation of both the left and right arms of right-handed subjects. To allow direct assessment of the lateralization of activation, left- and right-sided stimuli were delivered during separate PET scans. Both innocuous (35 degrees C) and painful (49 degrees C) stimuli were employed to determine whether lateralized processing occurred in a manner related to perceived pain intensity. Subjects were also scanned during a nonstimulated rest condition to characterize activation that was not related to perceived pain intensity. Pain intensity-dependent and -independent changes in activation were identified in separate multiple regression analyses. Regardless of the side of stimulation, pain intensity--dependent activation was localized to contralateral regions of the primary somatosensory cortex, secondary somatosensory cortex, insular cortex, and bilateral regions of the cerebellum, putamen, thalamus, anterior cingulate cortex, and frontal operculum. No hemispheric lateralization of pain intensity-dependent processing was detected. In sharp contrast, portions of the thalamus, inferior parietal cortex (BA 40), dorsolateral prefrontal cortex (BA 9/46), and dorsal frontal cortex (BA 6) exhibited right lateralized activation during both innocuous and painful stimulation, regardless of the side of stimulation. Thus components of information arising from the body surface are processed, in part, by right lateralized systems analogous to those that process auditory and visual spatial information arising from extrapersonal space. Such right lateralized processing can account for the left somatosensory neglect arising from injury to brain regions within the right cerebral hemisphere.

In vivo control of NMDA receptor transcript level in motoneurons by viral transduction of a short antisense gene.

Glutamate receptors play critical roles in normal and pathological processes. We developed an antisense gene delivery strategy to modulate the NMDA type of glutamate receptor. Using transient transfection in vitro and viral mediated gene transfer in vitro and in vivo, the effect of expression of an antisense gene fragment (60 bp) of the NR1 subunit was tested. Immunoblot analysis showed an antisense-concentration-dependent reduction in the NR1 subunit upon transient co-transfection of a plasmid expressing a sense NR1 gene and a plasmid expressing the antisense fragment into COS-7 cells. After recombination into an adenoviral vector, this antisense fragment reduced the amount of endogenous NR1 protein in PC12 cells. Finally, direct intraparenchymal injection of the viral vector into rat spinal cord resulted in diminished NR1 in motor neurons. Our results demonstrate the efficacy of this approach, which combines antisense with viral gene delivery to control the expression of specific genes in vivo. This approach may also be useful in reducing excitatory neurotransmission in vivo, with implications for the treatment of spinal disorders such as amyotrophic lateral sclerosis or chronic pain.

Anandamide activates vanilloid receptor 1 (VR1) at acidic pH in dorsal root ganglia neurons and cells ectopically expressing VR1.

The vanilloid receptor type 1 (VR1) is a heat-activated ionophore preferentially expressed in nociceptive neurons of trigeminal and dorsal root ganglia (DRG). VR1, which binds and is activated by capsaicin and other vanilloid compounds, was noted to interact with the endocannabinoid anandamide (ANA) and certain inflammatory metabolites of arachidonic acid in a pH-dependent manner. At pH < or = 6.5 ANA induced (45)Ca(2+) uptake either in primary cultures of DRG neurons or cells ectopically expressing C-terminally tagged recombinant forms of VR1 with an EC(50) = approximately 10 microm at pH 5.5. Capsazepine, a potent antagonist of vanilloids, inhibited ANA-induced Ca(2+) transport in both cell systems. Vanilloids displaced [(3)H]ANA in VR1-expressing cells, suggesting competition for binding to VR1. Ratiometric determination of intracellular free calcium and confocal imaging of the VR1-green fluorescent fusion protein revealed that, at low pH (< or =6.5), ANA could induce an elevation of intracellular free Ca(2+) and consequent intracellular membrane changes in DRG neurons or transfected cells expressing VR1. These actions of ANA were similar to the effects determined previously for vanilloids. The ligand-induced changes in Ca(2+) at pH < or = 6.5 are consistent with the idea that ANA and other eicosanoids act as endogenous ligands of VR1 in a conditional fashion in vivo. The pH dependence suggests that tissue acidification in inflammation, ischemia, or traumatic injury can sensitize VR1 to eicosanoids and transduce pain from the periphery.

Analysis of the native quaternary structure of vanilloid receptor 1.

Vanilloid receptor subtype 1 (VR1) is a ligand-gated channel that can be activated by capsaicin and other vanilloids as well as by protons and heat. In the present study, we have analyzed the oligomeric state of VR1. Co-immunoprecipitation of differently tagged VR1 molecules indicated that VR1 can form oligomers. Using two different heterologous VR1 expression systems as well as endogenous VR1 expressed in dorsal root ganglion cells, we analyzed oligomer formation using perfluoro-octanoic acid polyacrylamide gel electrophoresis. Results were confirmed both with chemical cross-linking agents as well as through endogenous cross-linking mediated by transglutaminase. Our results clearly show that VR1 forms multimers in each of the expression systems with a homotetramer as a predominant form. The oligomeric structure of VR1 may contribute to the complexity of VR1 pharmacology. Finally, differences in glycosylation between the systems were observed, indicating the need for caution in the use of the heterologous expression systems for analysis of VR1 properties.

Neuropeptide Y in trigeminal ganglion following chronic constriction injury of the rat infraorbital nerve: is there correlation to somatosensory parameters?

The aim of this study was to investigate neuropeptide Y (NPY) levels in trigeminal ganglia following infraorbital nerve injury. Two experimental procedures were performed in three groups of rats: a unilateral chronic constriction injury (CCI) to the infraorbital nerve (n=13), nerve manipulation without CCI (n=13) and unoperated controls (n=8). All rats underwent baseline and regular assessment of mechanical withdrawal threshold (Von Frey) and reaction to pin prick as well as free behavior evaluations. CCI to the infraorbital nerve induced significant hyperalgesia and allodynia within 9-12 days. At 6 days seven rats were euthanized and trigeminal ganglia harvested for immunocytochemical (ICC) studies. The study was ended at 14 days when all rats were euthanized and their ganglia harvested for ICC and radioimmunoassay (RIA) studies. An increase in NPY levels was seen in the ipsilateral ganglia of manipulated and CCI rats at 6 days, when rats displayed no pain-related behavior. At 14 days, ICC and RIA both detected significant increases in NPY levels in the ipsilateral ganglia of CCI and manipulated rats but not in unoperated controls. The possible roles of NPY in pain modulation and nerve injury are discussed in light of these findings.

Spinal cord genes enriched in rat dorsal horn and induced by noxious stimulation identified by subtraction cloning and differential hybridization.

Persistent nociceptive input increases neuronal excitability and induces a program of gene expression in the dorsal spinal cord. The alteration in gene expression commences with phosphorylation and induction of immediate early genes and proceeds to target genes. Only a few target genes have been identified as yet. The present report uses a polymerase chain reaction-based subtraction cloning procedure to obtain an "anatomically focused" complementary DNA library enriched in transcripts related to sensory spinal cord (rat dorsal horn minus ventral horn). A subset of clones from this library (n=158) was screened to verify dorsal horn enrichment and to identify those regulated by carrageenan-induced peripheral inflammation. Molecular classes which displayed enriched expression included a proto-oncogene not previously associated with sensory processes, two regulators of the Rho/Rac pathway which controls cell shape, and three genes involved in cytoskeletal regulation and scaffolding. Additional transcripts coded for proteins involved in intercellular communication or intracellular function. Within the set of 158 transcripts, one known and two unknown genes were induced by persistent noxious input. The known gene codes for the secreted cysteine proteinase inhibitor, cystatin C, suggesting that modulation of extracellular proteolytic activity occurs. Since it is secreted, cystatin C may also provide a cerebrospinal fluid bio-marker for persistent pain states. Using a combined anatomical and functional approach, we have extended the molecular repertoire of genes expressed and induced in second-order neurons or supporting glial cells in several new directions, with particular emphasis on regulation of cell morphology and plasma membrane dynamics. Some of these proteins reveal new pathways for information signaling in the sensory half of the spinal cord and require further research to understand their role in the adult spinal cord. The induced genes may provide new molecular targets for therapeutic development and provide new probes for investigating the dynamic state of cellular activity that occurs during persistent pain states.

Ligand-induced dynamic membrane changes and cell deletion conferred by vanilloid receptor 1.

The real time dynamics of vanilloid-induced cytotoxicity and the specific deletion of nociceptive neurons expressing the wild-type vanilloid receptor (VR1) were investigated. VR1 was C-terminally tagged with either the 27-kDa enhanced green fluorescent protein (eGFP) or a 12-amino acid epsilon-epitope. Upon exposure to resiniferatoxin, VR1eGFP- or VR1epsilon-expressing cells exhibited pharmacological responses similar to those of cells expressing the untagged VR1. Within seconds of vanilloid exposure, the intracellular free calcium ([Ca(2+)](i)) was elevated in cells expressing VR1. A functional pool of VR1 also was localized to the endoplasmic reticulum that, in the absence of extracellular calcium, also was capable of releasing calcium upon agonist treatment. Confocal imaging disclosed that resiniferatoxin treatment induced vesiculation of the mitochondria and the endoplasmic reticulum ( approximately 1 min), nuclear membrane disruption (5-10 min), and cell lysis (1-2 h). Nociceptive primary sensory neurons endogenously express VR1, and resiniferatoxin treatment induced a sudden increase in [Ca(2+)](i) and mitochondrial disruption which was cell-selective, as glia and non-VR1-expressing neurons were unaffected. Early hallmarks of cytotoxicity were followed by specific deletion of VR1-expressing cells. These data demonstrate that vanilloids disrupt vital organelles within the cell body and, if administered to sensory ganglia, may be employed to rapidly and selectively delete nociceptive neurons.

Intrathecally administered cholera toxin blocks allodynia and hyperalgesia in persistent pain models.

In persistent pain, the spinal cord concentration of the opioid peptide dynorphin increases dramatically, yet the function of dynorphin remains unknown. If prodynorphin expression could be manipulated in vivo, it might be possible to determine what role dynorphin plays in persistent pain. Previous work in our laboratory showed that prodynorphin expression is regulated through the cyclic adenosine monophosphate pathway. Therefore, we attempted to enhance prodynorphin expression in the spinal cord of rats by stimulating adenylate cyclase with cholera toxin; however, contrary to our hypothesis, intrathecally administered cholera toxin did not enhance prodynorphin expression. Rather, cholera toxin suppressed the increase in prodynorphin produced by inflammation. Cholera toxin also inhibited the allodynia and hyperalgesia associated with inflammation and nerve injury. Interestingly, the antiallodynic and antihyperalgesic actions of cholera toxin were reversed with the opioid receptor antagonist, naloxone. These findings suggest that cholera toxin enhances or unmasks an endogenous opioid pathway to produce its antiallodynic and antihyperalgesic effects. Furthermore, these data indicate that the suppression of the inflammation-induced increase in spinal cord prodynorphin is caused by the opioid-mediated decrease in the nociceptive stimulus.

Co-localization of N-methyl-D-aspartate receptors and substance P (neurokinin-1) receptors in rat spinal cord.

Glutamate, substance P (SP), and their receptors have been implicated in the initiation and maintenance of persistent pain through an interaction at second order spinal cord neurons. Employing well-characterized antibodies to the SP receptor and the N-methyl-D-aspartate receptor (NR1 subunit, splice variant missing exon 22), we demonstrate co-localization of these receptors on second order neurons at cervical, thoracic, lumbar, and sacral spinal cord levels. The co-localization was marked in lamina I of the dorsal horn at all levels and in the intermediolateral nucleus of the thoraco-lumbar spinal cord nuclei associated with autonomic function.

Chronic tooth pulp inflammation causes transient and persistent expression of Fos in dynorphin-rich regions of rat brainstem.

We have analyzed central Fos immunoreactivity (Fos-IR) brainstems of adult rats after three clinically relevant dental injuries: filled dentin (DF) cavities that cause mild pulp injury and heal within 1-2 weeks; open pulp exposures (PX) that cause gradual pulp loss and subsequent periodontal lesions; and filled pulp exposures (PXF). By 1 week after DF cavities, no Fos-IR remained except for sites such as lateral-ventral periolivary nucleus (LVPO) that had Fos-IR in all rats including controls. PX injury induced (1) a delayed transient expression of Fos at 1-2 weeks at three loci (ipsilateral neurons in dorsomedial nucleus oralis, paratrigeminal nucleus, and trigeminal tract), (2) persistent ipsilateral Fos for at least 4 weeks after injury in dynorphin (Dyn)-rich regions (rostral lateral solitary nucleus, periobex dorsal nucleus caudalis), and (3) late Fos-IR at 2-4 weeks (bilateral superficial cervical dorsal horn, contralateral dorsal nucleus caudalis, contralateral rostral lateral solitary nucleus). Rats with PXF injury were examined at 2 weeks, and they had greater numbers and more extensive rostro-caudal distribution of Fos neurons than the PX group. One week after PX injury, Fos-IR neurons were found in regions with strong Dyn-IR central fibers. Co-expression of Dyn and Fos was found in some unusually large neurons of the ipsilateral rostral lateral solitary nucleus, trigeminal tract, and dorsal nucleus caudalis. Immunocytochemistry for the p75 low affinity neurotrophin receptor (p75NTR) or for calcitonin gene-related peptide (CGRP) showed no consistent change in trigeminal central endings in any Fos-reactive brainstem areas, despite the extensive structural and cytochemical reorganization of the peripheral endings of the dental neurons. The Fos responses of central neurons to tooth injury have some unusual temporal and spatial patterns in adult rats compared to other trigeminal injury models.

Genomic integration and gene expression by a modified adenoviral vector.

A replication-deficient recombinant adenovirus encoding luciferase was constructed using 5' and 3' long terminal repeat (LTR) sequences of the Moloney murine leukemia virus. Gene expression was observed in cultured cells in vitro and in submandibular gland, cortex, and caudate nucleus for as long as three months in vivo. The vector integrated randomly into the genome of both dividing and nondividing cells as determined by fluorescence in situ hybridization (FISH) (10-15% of cells in vitro and 5% in rat spleen in vivo), gene walking, Southern hybridization, and polymerase chain reaction (PCR), in the absence of transcomplementing reverse transcriptase or integrase activity. The new vector combines the high titer and versatility of adenoviral vectors with the long-term gene expression and integration of retroviral vectors.

Localization of alternatively spliced NMDAR1 glutamate receptor isoforms in rat striatal neurons.

Alternative splicing of the mRNA encoding the N-methyl-D-aspartate (NMDA) receptor subunit NR1 changes the structural, physiologic, and pharmacologic properties of the resultant NMDA receptors. We used dual label immunocytochemistry and confocal microscopy to localize the four alternatively spliced segments of the NR1 subunit (N1, C1, C2, and C2') in rat striatal neurons. Striatofugal projection neurons and four populations of interneurons were studied. Projection neurons, which were identified by immunolabeling for calbindin and by retrograde tracing from the globus pallidus and the substantia nigra, were the only striatal neurons containing C1 segment immunoreactivity. Projection neurons were also C2 segment immunopositive, as were all other neuronal populations studied. Projection neurons were C2' segment immunonegative. In contrast, each of the interneuron types were labeled by the antibody to the C2' segment: nitric oxide synthase interneurons were labeled intensely, calretinin and parvalbumin neurons were labeled moderately strongly, and cholinergic neurons were also labeled but less strongly than the other types of interneurons. Parvalbumin interneurons showed distinct N1 segment immunolabeling, which was not found in other types of striatal neurons. Our results suggest that all striatal neurons studied synthesize NR1 subunit proteins, but the isoforms of the protein present in projection neurons and the several types of interneurons are distinct. This differential expression of NR1 isoforms may affect both neuronal function and selective vulnerability of neurons to injury.

Pain intensity processing within the human brain: a bilateral, distributed mechanism.

Functional imaging studies of human subjects have identified a diverse assortment of brain areas that are engaged in the processing of pain. Although many of these brain areas are highly interconnected and are engaged in multiple processing roles, each area has been typically considered in isolation. Accordingly, little attention has been given to the global functional organization of brain mechanisms mediating pain processing. In the present investigation, we have combined positron emission tomography with psychophysical assessment of graded painful stimuli to better characterize the multiregional organization of supraspinal pain processing mechanisms and to identify a brain mechanism subserving the processing of pain intensity. Multiple regression analysis revealed statistically reliable relationships between perceived pain intensity and activation of a functionally diverse group of brain regions, including those important in sensation, motor control, affect, and attention. Pain intensity-related activation occurred bilaterally in the cerebellum, putamen, thalamus, insula, anterior cingulate cortex, and secondary somatosensory cortex, contralaterally in the primary somatosensory cortex and supplementary motor area, and ipsilaterally in the ventral premotor area. These results confirm the existence of a highly distributed, bilateral supraspinal mechanism engaged in the processing of pain intensity. The conservation of pain intensity information across multiple, functionally distinct brain areas contrasts sharply with traditional views that sensory-discriminative processing of pain is confined within the somatosensory cortex and can account for the preservation of conscious awareness of pain intensity after extensive cerebral cortical lesions.

Epidural resiniferatoxin induced prolonged regional analgesia to pain.

Adequate treatment of cancer pain remains a significant clinical problem. To reduce side effects of treatment, intrathecal and epidural routes of administration have been used where appropriate to reduce the total dose of agent administered while achieving regional control. Resiniferatoxin (RTX), an ultrapotent capsaicin analog, gives long-term desensitization of nociception via C-fiber sensory neurons. We evaluate here the analgesic effect on rats of epidurally administered RTX, using latency of response to a thermal stimulus in unrestrained animals. Results were compared with those for systemically administered RTX. Vehicle or graded doses of RTX were injected subcutaneously (s.c.) or through an indwelling lumbar (L4) epidural catheter as a single dose. Both routes of application of RTX produced profound thermal analgesia, reaching a plateau within 4-6 h and showing no restoration of pain sensitivity over 7 days. Vehicle was without effect. For the epidural route, the effect was selective as expected for the targeted spinal cord region, whereas the subcutaneous administration of RTX had a generalized analgesic effect. At doses yielding a tripling of back paw withdrawal latency, epidural treatment was 25-fold more effective than the subcutaneous route of application. Consistent with the regional selectivity of the lumbar epidural route, the front paws showed no more effect than by systemic RTX treatment. Binding experiments with [3H]RTX provided further evidence of the segmental desensitization induced by epidural RTX. We conclude that epidural administration of RTX at the lumbar spinal level produces profound, long-lasting, segmental analgesia to C-fiber mediated pain in the rat.