Neuroimaging of Pain: Advances and Future Prospects

Neuroimaging of Pain: Advances and Future Prospects

The Journal of Pain, Vol 9, No 7 (July), 2008: pp 567-579 Available online at www.sciencedirect.com Critical Review Neuroimaging of Pain: Advances an...

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The Journal of Pain, Vol 9, No 7 (July), 2008: pp 567-579 Available online at www.sciencedirect.com

Critical Review Neuroimaging of Pain: Advances and Future Prospects Diane T. Stephenson* and Stephen P. Arneric† *Pfizer Global Research and Development, Groton, Connecticut, and † Neuromed Pharmaceuticals Ltd., Vancouver, BC, Canada

Abstract: Chronic pain conditions remain a high unmet medical need, and a significant number of patients are not effectively treated with currently available therapies. There is a significant challenge in developing more effective therapies to treat pain, particularly in chronic debilitating pain conditions such as neuropathic pain. Preclinical research has been beneficial in advancing mechanistic understanding of the pathophysiology of pain as well as in defining new therapeutic targets for intervention. However, the increased understanding of the neurobiology of pain has not yet translated into breakthroughs in pain therapies. Some debate exists as to how predictive the common animal models of pain are to the human condition. Translation animal model activity promises to be enhanced by application of novel neuroimaging technologies. It is well acknowledged throughout the industry that the application of preclinical to clinical translational biomarkers is an important strategy that holds promise in increasing the confidence in the translatability of the preclinical to clinical data. Imaging biomarkers have tremendous potential for affecting pain research from both diagnostic as well as therapeutic standpoints. Noninvasive imaging has the inherent advantage of being able to evaluate central mechanisms of pain and the effects of intervention both in animals and in humans. Because each subject serves as its own control, the inherent intersubject variabilities can be less of a confound. This review discusses both the promise and limitations of using imaging modalities to study pain processing and integrates it into the evolving drug discovery and development paradigm. Each section summarizes current clinical reports and, if applicable, preclinical translational findings. Emphasis is given to technical areas for future development and revealing neuroinflammation dynamics and targets that are influenced by genetics and cellular insults. With continued application of neuroimaging technologies, new therapeutic approaches to treat chronic pain as well as define tools to assess functional outcomes promise to emerge. Perspective: This review discusses the promises and limitations of using noninvasive imaging modalities to study pain processing and integrates it into the evolving drug discovery and development paradigm. Emerging neuroimaging technologies may spawn new therapeutic approaches to treat chronic pain as well as define translational tools to assess functional clinical outcomes. © 2008 by the American Pain Society Key words: Neuroimaging, neuropathic pain, neuroinflammation, PET, SPECT, MRI, neuroplasticity, apoptosis, optical imaging.

I Supported by Pfizer and Neuromed Pharmaceuticals, Inc. Address reprint requests to Dr. Diane Stephenson, Pfizer Global Research and Development, Eastern Point Road, MS8220-4224, Groton, CT 06340. E-mail: [email protected] 1526-5900/$34.00 © 2008 by the American Pain Society doi:10.1016/j.jpain.2008.02.008

t is well acknowledged throughout the industry that the application of preclinical to clinical translational biomarkers is an important strategy that holds promise in increasing the confidence in the translatability of the preclinical to clinical data. There are many different animal models, primarily carried out in rodents, that are used to mimic the various forms of pain. Animal models have been very useful in illuminating the putative mechanisms by which chronic pain develops and is maintained.16,85 567

568 Imaging biomarkers have tremendous potential for affecting pain research from both diagnostic as well as therapeutic standpoints.43,66 Advances in imaging technologies in recent years have progressed at record pace. Imaging that was possible 5 years ago has been revolutionized with advances in clinical and preclinical imaging technologies and instrumentation. Single imaging methods have now been combined into multimodal imaging approaches such as the positron emission tomography (PET)/volumetric CT unit, which allows coregistration of functional with anatomic data. These and future new techniques have tremendous utility in a wide variety of applications to visualize the onset and progression of disease processes in individual subjects over time and are becoming more common in the fields of cardiology and oncology.118,120 Dual modality instruments are also being developed for preclinical applications.50 The identification and validation of imaging biomarkers for chronic pain have lagged behind areas such as oncology, cardiovascular medicine, and neurology. For some time now, industry-wide consortia existed in partnership with academia as a beneficial strategy in helping validate imaging tools, enable technology development, and spread the risks and costs among key stake holders (eg, the Alzheimer’s disease [AD] neuroimaging initiative, http://www.nia.nih.gov/Alzheimers/ResearchInformation/ClinicalTrials/ADNI.htm; and the Osteoarthritis initiative, http://www.oai.ucsf.edu/datarelease/). More recently, the Imaging Consortium for Drug Development (ICD, https://meitner.mclean.harvard.edu/) is underway, with a focus on functional magnetic resonance imaging (fMRI) to elucidate the effects of pain modulating drugs on neural activity within the brain in humans and in animal models of central nervous system (CNS) disease. Establishment of ICD is an important catalyst to realize the benefits of standardization and cost-sharing among consortium members for precompetitive neuroscience research. Neuroimaging provides a “window into the brain” that can be used to unravel the systems dynamics as well as the progression of pain states. Classic methods to study the mechanisms of nociception, including neuroanatomic, neurophysiological, biochemical, behavioral, and molecular approaches, have helped advance the understanding of many pathophysiologic processes involved in pain and pain perception. One key advantage that makes imaging particularly appealing is the inherent ability of the technology to monitor temporally, in the same subject, key functional and physiologic processes under dynamic conditions. In this way, intersubject variability is reduced since each subject serves as its own control and can be monitored before and after treatment. Recent progress in imaging technology has provided a new impetus to investigate multiple dimensions of pain. Another particularly appealing feature of imaging is that the technology can bridge from animals to humans. In several instances, clinical imaging tools are further advanced in technology than those developed for preclinical purposes. However, the ability to apply imaging technologies in both animals and humans is a critical

Neuroimaging of Pain: Advances and Future Prospects success factor in translational research and facilitates the development of mechanistic and outcome translational biomarkers. This review discusses both the promise and limitations of using imaging modalities to study pain processing and integrates it into the evolving drug discovery and development paradigm. Each section summarizes current clinical reports and, if applicable, preclinical translational findings. Emphasis is given to technical areas for future development and revealing targets of cellular plasticity influenced by genetics and cellular insults. With continued application of neuroimaging technologies, new therapeutic approaches to treat chronic pain as well as define tools to assess functional outcomes promise to emerge.

Imaging Modalities Positron Emission Tomography PET can be used as a measure of local brain activity by using radionuclides to produce maps representing changes in cerebral blood flow. PET has been applied to investigate the neural substrates involved in pain processing and perception in human subjects. Regional cerebral blood flow, an indirect index of synaptic neuronal activity, can be measured directly by assessing changes in uptake of H215O, inhaled C15O2, or 15O-butanol.54 Such methods have been applied to investigation of the neuroanatomic substrates of chronic ongoing neuropathic pain51 and experimentally induced acute pain.18,57 The higher-order central neuroanatomic substrates of pain have been illuminated by using PET and other functional imaging approaches. Cortical regions involved in processing of painful stimuli include primary somatosensory cortex, secondary somatosensory cortex, parietal operculum, insula, anterior cingulate cortex, and prefrontal cortex.110,116 Such data suggest that the brain uses different or unique central mechanisms for chronic pain as compared with experimental acute pain. In patients with neuropathic pain, PET imaging of regional blood flow demonstrated that activity in the cortical network involved in the sensory-discriminative processing of nociceptive pain is increased in neuropathic pain while decreased activity occurs in the orbitofrontal and insular cortices.131 Furthermore, different components of pain affect can be imaged simultaneously by using PET. Cortical areas involved in perception of pain have been illuminated by applying the same intensity of painful stimuli while varying the unpleasantness of noxious stimuli by hypnosis.97 PET revealed significant changes in activity in anterior cingulate gyrus, whereas no alterations were observed in the primary somatosensory cortex, suggesting limbic frontal lobe activity mediates pain perception.97 In subcortical brain centers, cerebral blood flow of higher-order brain centers mediating chronic pain revealed a reduction in the thalamus contralateral to the body regions affected by neuropathic pain.53 Such functional alterations in thalamic pain processing circuits may represent altered modulation that is intrinsically linked to the pathophysiological changes accompanying neuro-

Stephenson and Arneric pathic pain. This apparent uncoupling during chronic pain (perhaps due to a loss of local inhibitory circuits as seen in the dorsal horn125) may serve as a biomarker for some types of neuropathic pain. Interestingly, the perceived intensity of experimental pain is strongly associated with the magnitude of PET signals across multiple brain regions.30,113,21 These studies suggest that there is a significant relationship between perceived pain intensity and activation of functionally diverse brain regions making PET as a prime technology for assessing responses to therapeutic intervention. PET has been applied to study the effect of analgesic treatments in human subjects. Changes in cerebral blood flow correlate with pain relief in neuropathic pain patients subjected to motor cortex stimulation.92 PET has also been a powerful tool to elucidate potential relationships between chronic pain states and the dopaminergic system. For example, presynaptic dopaminergic activity in fibromyalgia patients is reduced.132 In patients with Parkinson’s disease, increased pain thresholds have been reported after levodopa administration.14 Thus, imaging of the dopaminergic system in patients with functional pain disorders suggests attenuation of dopaminergic activity may underlie some chronic pain states. More recently, the disrupted dopaminergic reactivity in fibromyalgia patients has been suggested to be a critical factor underlying the widespread pain and discomfort in fibromyalgia133 and suggests that the therapeutic effects of dopaminergic treatments for this intractable disorder should be explored.134 Given the relationship between stress and dopamine, these approaches have relevance for unraveling the potential relationship between chronic pain states and stress.132 Applications of PET also include neuroimaging using specific radioligands for evaluation of receptor occupancy and density. Ligand PET studies allow evaluation of receptor distribution under control and disease states. With receptor occupancy, a well-characterized imaging radioligand is evaluated in combination with a selective investigational receptor agonist or antagonist competing for the same sites in vivo; it is possible to demonstrate that the levels of radioactivity in target tissues should be reduced in a dose-dependent fashion and reflect increased receptor occupancy by the candidate drug. It is possible then to address the percent receptor occupancy sufficient for mediating functional improvement in outcome.61,38 Imaging of opioid receptors has been particularly illuminating in pain research. Two opioid receptor radiotracers, the ␮-opioidergic agonist 11C-carfentanil and the nonselective opioid receptor antagonist 11C-diprenorpine, have been applied in studies of human pain conditions.105 11C-carfentanil neuroimaging demonstrated that sustained pain triggers release of endogenous opioids in region specific manner and that a reduction of severity of pain correlates with ␮-opioid receptor activation.143 Evaluation of opioid receptors using 11Cdiprenorphine reveals reduced density of opioid receptors in patients with central neuropathic pain resulting from poststroke pain syndrome58,129 but not in conditions of peripheral neuropathic pain.69 Imaging of opi-

569 oid receptors in vivo thus illuminates the value of PET imaging in unraveling the complexities of different pain states. Novel radioligands are being developed for evaluation of pain conditions including novel opioid receptor tracers63 and ␴-receptor tracers.22 Ligand PET studies have direct application and translatability in preclinical studies of pain. Ex vivo receptor occupancy, autoradiographic radioreceptor distribution, and in vivo micro-PET studies in preclinical models of pain represent investigational tools directly applicable to PET neuroimaging of pain in humans. Regional blood flow assessment in animals has illuminated the translatable neuroanatomic regions in rats that parallel those regions activated in humans using PET imaging. The hallmark technique of 2-deoxyglucose (2DG) utilization is a highly sensitive and well-established method for assessing local changes in cerebral glucose consumption in small animals.104 Application of 2-DG to rodent models of pain has shown metabolic changes in spinal cord after noxious stimuli in a rats subjected to spinal cord transection,20 in brain and spinal cord of rats after chronic constrictive nerve injury,74 and in the spinal cord of rats subjected to formalin-induced noxious stimulation.96 In all cases, the functional activity pattern as revealed with quantitative 2-DG autoradiography varied according to the time and changes in animal behavior. Heterogeneous rates of glucose consumption have been reported to correlate with noninvasive assessment of cerebral glucose utilization such as FDG-PET,9 making it a suitable translational research tool between rodents and humans. The most significant limitation of this technique is that it is slow and labor-intensive. However, with new progress in automated image analysis methods, there is hope that enabling technology advances will make it more efficient in the future.

Single Photon Emission Computerized Tomography Radioligands for single photon emission computerized tomography (SPECT) have similar potential as PET radioligands for molecular imaging of particular neurotransmitter receptors as well as assessment of regional cerebral blood flow in vivo. Like PET, SPECT is based on recording emissions from injected radionuclides; however, the isotopes emit single photons of lower energy. Although the cost of SPECT is less, the disadvantages include poorer spatial resolution and slow clearance of radiochemicals. SPECT can also be used to measure regional cerebral blood flow. Specific radionuclides used in SPECT imaging include lipophilic agents that readily cross the bloodbrain barrier and then become trapped in the CNS, thus allowing measurement of cerebral blood flow. In human patients with chronic pain, the tracer technetium-99m hexamethylpropylene amine oxime (99mTe-HMPAO) demonstrates reduced regional blood flow in thalamus.83 In patients undergoing spinal cord stimulation for treatment of chronic pain, the regional cerebral blood flow response assessed by SPECT was found to vary ac-

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cording to the response to treatment. These data highlight the suitability of SPECT in imaging of pain as well as monitoring patient response to therapy. Applications of SPECT have been more limited than PET in studies of pain in animals and humans. For example, there are no opioid receptor SPECT ligands that have been applied to humans at the present time. In baboons, the diprenorphine analogue C6-[123I] iodoallyldiprenorphine shows specific radioligand binding to predicted brain regions by SPECT imaging,64 and several candidate ligands have been pursued in rodents, particularly on ligands for the ␦-opioid receptor.63 In animals, patterns of activation in rat forebrain revealed by SPECT, particularly limbic system pathways, were activated during prolonged and persistent pain induced by subcutaneous administration of formalin.80 Corresponding data are not available in humans.

Functional Magnetic Resonance Imaging Functional magnetic resonance imaging (fMRI) is a powerful noninvasive tool for imaging functionally active brain regions in health and disease. The most common method is blood oxygenation level-dependent (BOLD) imaging. fMRI BOLD imaging is based on the magnetization difference between oxyhemoglobin and deoxyhemoglobin, which constitutes a measure of hemodynamic response. The BOLD contrast is thought to reflect the population synaptic activity of given brain regions. A combination of fMRI with drug administration, termed pharmacologic magnetic resonance imaging (phfMRI), holds tremendous promise for neuroimaging mechanistic studies and is ideally suited for testing the effects of drug intervention. Drug-induced changes in brain activity as well as pharmacologic modulation of task-induced activities are common applications of phfMRI. Key examples highlighting the potential applicability of phfMRI applications for drug testing include evaluation direct of effects of drug on CNS patterns of activity, effects of drugs on task-related activation, chronic versus acute effects of drugs, side effect profiles of novel drugs, effects on cerebral metabolism, and drug-induced changes in different patient populations.12,28,76,99,130 The critical assumption in fMRI/phfMRI studies of pain is that the nociceptive or pain signal is manifested as an increase in metabolic activity evoking an increase in blood flow, increased cerebral blood volume, decreased deoxyhemoglobin, and thereby an increased magnetic resonance imaging (MRI) signal. fMRI of pain has produced results that are in overwhelming agreement with those reported using PET.6,29 In addition, fMRI has illuminated novel regions of the brain involved in pain processing such as the nucleus of the solitary tract.138 In patients with neuropathic pain, fMRI was used to delineate a comprehensive inventory of brain regions involved in response to allodynic stimuli.92,93 High-resolution fMRI has been applied to imaging of the brainstem in human subjects exposed to painful stimuli.32 In the spinal cord, fMRI can be applied to measure neuronal activity in both healthy and injured human subjects.109 After application of cold noxious stimuli, activa-

tion has been observed in the ipsilateral dorsal horn with activity changes corresponding to the stimulus intensities; activations were notably attenuated in patients with spinal cord injuries.109 fMRI has also been applied to investigate pain-related changes in second-order neuroanatomic regions. As has been observed with PET, fMRI demonstrates that the magnitude of the fMRI signal reflects the perceived intensity of pain.10,96,101,126 Interestingly, different types of pain have been associated with different patterns of brain activation. For example, thermal and mechanical hyperalgesia produce substantially different brain activation patterns.70 The neuroanatomic complexity of pain processing with functionally related neural systems (eg, emotive) can be visualized with fMRI. The brain responds both before and during the application of noxious stimuli. fMRI has also revealed that particular anatomic regions, such as the periaqueductal gray (PAG), are activated during the anticipatory phases of processing noxious stimuli.36 It has been suggested that the anticipatory response to pain may modulate the perception and maintenance of chronic pain.59,96 In fact, subjects who report strong anticipation before painful stimulation experience more intense pain, and this link between anticipation and perception of pain is associated with increased activation in the ventral tegmental area (VTA), PAG, and entorhinal cortex.36 In patients with chronic pain, the expectation of pain is thought to predict pain perception.95 Pain perception can be increased through high anxiety95 or decreased through stress-induced analgesia.37 Directed attention to painful stimuli is associated with increased synchronization of neuronal activity in the pain cortical network as assessed with electrophysiologic measures.87 These data are consistent with neuroimaging studies demonstrating distinct patterns of activation in the anticipatory versus the processing phases of pain. Not surprisingly, anxiety and depressive disorders also accompany chronic pain states. However, fMRI activation patterns in the amygdala can, in fact, be sufficiently differentiated between fibromyalgia patients with and without depression.42 Inter-relationships between cognitive function and pain have been investigated by using fMRI. For example, painrelated activity in multiple cortical areas is modulated by execution of an attention-demanding task.4,91 Interestingly, the complexity of the cognitive task has an impact on the subjective pain rating as well as neuroanatomic regions activated in human subjects.126 Such investigations have suggested that interventions directed at the attentional aspects of pain may be a viable strategy to treatment of chronic pain conditions. Thus, neuroimaging substantiates that both emotional and cognitive mechanisms affect pain perception. fMRI has been applied to investigation of rodent models of pain. Functional activity has been assessed in the rat spinal cord in models of noxious electrical stimulation60 and capsaicin forepaw administration.72 Interestingly, the study of Lawrence et al60 demonstrated a correlation between neuronal activity assessed by fMRI with

Stephenson and Arneric that measured by histopathologic assessment of immediate early gene induction. In higher-order brain regions, fMRI activation delineates similar brain regions activated in rats and in humans subjected to capsaicin treatment; however, the direction of effect of mechanical stimulation on brain activation in PAG differed between awake humans and anesthetized rats. These differences may be due to species variations, effects of anesthesia, or as-yet undefined mechanisms. In summary, fMRI investigation provides novel information about higher-order brain centers involved in nociceptive processing in animals and humans. Relating neural activity changes to the varied pain experiences has led to an increased awareness of how factors (eg, cognition, emotion, context, injury) can separately influence pain perception. Tying this body of knowledge in humans to work in animal models of pain provides an opportunity to determine common features that reliably contribute to pain perception and its modulation.115

Diffusion Tensor Imaging Diffusion tensor imaging (DTI) is a magnetic resonance imaging– based method that can map white matter anatomical connections in the living human brain. Directionality of water movement is determined to illuminate the orientation of white matter tracts as well as enable visualization of functionally localized brain regions.23 DTI has been applied, in human subjects, to investigating white matter connections from the PAG and nucleus cuneiformis with regions involved in mediating pain.47 Such tractography studies support the top-down neuroanatomic pain processing pathways that modulate pain perception described earlier. Although the application of DTI to imaging of pain has been somewhat limited to date, the ability to map white matter tracts noninvasively within the human brain promises to be a powerful tool to define a functional connectivity among the variety of structures mediating complex pain syndromes. These pathways may become altered in pain states. One case study in a post-stroke pain subject combined DTI with fMRI and found that damage of thalamic nociceptive fibers was associated with release of activity in cortical regions.102 A combination of neuroimaging modalities holds promise in future studies to enable functional and structural assessments simultaneously.

Volumetric MRI Using volume-based morphometry, significant reductions have been seen in gray matter volume of the dorsolateral prefrontal cortex (dlPFC) and the right thalamus in patients with chronic back pain.2 The magnitude of the decrease is equivalent to the loss of gray matter volume in 10 to 20 years of normal aging. There was a correlation between loss of brain volume and duration of pain suggesting a 1.3-cm3 loss of gray matter volume for every year of chronic pain. The observed pattern of atrophy is distinct from that described in chronic depression or anxiety1,135 and conditions of neurodegenera-

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tion such as Alzheimer’s disease. It is not clear how universal this finding is to other forms of pain. This is clearly an emerging area. For example, no significant changes in brain volume were previously described in migraine.75 Yet, in a very recent study, a thickening of the somatosensory cortex has been reported in migraine patients.27 Further neuroimaging studies in multiple types of chronic pain syndromes will be necessary to explore this further. It is worth noting, however, that neurodegenerative mechanisms may explain the finding of volumetric brain loss in chronic pain. There are numerous reports of parallels between pain and neurodegeneration on a molecular level121 as well as in the context of pathology. Pathologic hallmarks shared between pain and neurodegeneration include neuroinflammation, glial activation, and apoptosis. Such similarities in pathologic processes suggest that neuroimaging approaches that are currently being applied to neurodegeneration may be successful in illuminating the underlying pathophysiology of chronic pain states. Findings that implicate neurodegenerative sequelae of chronic pain may lead to novel therapeutic strategies to both treat and monitor chronic pain outcomes. This is likely to be a changing landscape given the advances in higher resolution instrumentation and image analysis algorithms.

Magnetic Resonance Spectroscopy In vivo proton magnetic resonance spectroscopy is a noninvasive technique that allows the biochemical assessment of small molecules in precise anatomic regions of tissue. Identification of metabolic biomarkers is assessed by spectroscopic measurement within a volumetric region of interest. In the brain, magnetic resonance spectroscopy (MRS) is commonly used to measure N-acetylaspartate (NAA), a marker of neuronal somata and axons and myoinositol, which is thought to be produced in glia. NAA levels represent an indicator of changes in neuronal viability and function and are known to be reduced in pathologic conditions such as Alzheimer’s disease.90 Similarly, NAA levels change in the dlPFC in chronic back pain subjects.45 Furthermore, thalamic NAA levels are reduced in neuropathic pain patients as compared with control subjects, and NAA levels were most dramatically reduced in patients who did not respond to conventional pain treatment.40 These data support the volumetric changes described above and suggest that MRS may be an additional useful imaging modality to explore the relationship between neurodegeneration and chronic pain.

Neuroimaging Challenges and Limitations The complexity of pain includes the fact that it has many different dimensions of central representation in addition to the direct sensory component. Other aspects include emotional, anticipatory, affective, and cognitive components. These complexities make it challenging to assign which particular imaging phenomenon corresponds to which specific submodality being visualized or

572 measured. Disparate findings are observed in brain imaging studies of experimentally induced pain in human subjects. This is likely due to the complexity of central mechanisms that underlie the matrix of experiences involved in mediating pain in humans. Additional complexities inherent to the experimental model systems also play a role. For example, it has been suggested that lying in an imaging scanner while experiencing pain requires active suppression of motor commands for withdrawal from the painful stimulus; alternatively, a sensation of novel exposure to the scanner environment (eg, claustrophobia) can elicit unique brain activation patterns independent of pain processing and introduce significant interpatient variability as well as intersession variability that decreases statistical power. Some have cautioned interpretation of models that involve tactile pain stimuli, since the brain mechanisms involved in mechanical, pressure, and tactile processing may be independent of pain processing. To overcome such confounds, noncontact computer-controlled laser stimulation that precisely administers painful stimuli have been studied using neuroimaging methods.137 The CO2 laser delivers energy at a wavelength with sufficient depth of penetration to activate nociceptors without causing skin damage. fMRI of human subjects with this stimulation paradigm revealed unique attributes of lateralization in both thalamus and cortex.137 An additional complication in evaluating neuroimaging data from the pain literature is that a wide variety of differences exist in terms of the specific anatomic regions that are engaged in chronic pain states. This is most likely attributable to a large number of factors such as differences in the degrees and levels of pain or injury, the type of pain, as well as the overall condition of the patient. Interindividual differences in pain somatotopy may exist. Heterogeneous results in imaging outcomes between subjects may reflect underlying differences in the pathophysiologic pain processes between subjects or may also be due to imaging differences from a technical perspective (types of scanners, multi-imaging modalities). There is also evidence to suggest that individuals may have different thresholds to painful stimuli. An elegant study by Coghill et al19 compared psychophysical readings to define pain sensitivity with fMRI BOLD imaging to assess brain activity. It was reported that highly sensitive individuals exhibit greater activation of the primary somatosensory cortex, anterior cingulate cortex, and the prefrontal cortex as compared with insensitive subjects. There is a need to assess large numbers of patients to specifically investigate the influence of clinical variables on brain activation patterns. From an instrumentation point of view, there are substantial technical difficulties in applying functional imaging to brainstem and spinal cord, the areas of most robust pathology. Challenges include poor spatial resolution, image distortion, and signal losses due to inhomogeneity.114 Additional concerns include the consistency of acquisition and data analysis approaches and the critical importance of performing validation studies such as test-retest reliability.

Neuroimaging of Pain: Advances and Future Prospects Adjustment for movement-related artifacts in fMRI studies has been a focus of attention.39 Recent technologic advances have facilitated imaging of very small regions. For example, it has recently been demonstrated that volumetric MRI of dorsal root ganglia can be applied to quantify neuronal death after peripheral nerve injury.124 Such advances will facilitate even greater ability to resolve very subtle pathologies in vivo as applied to pain. Preclinically, there is still debate as to the predictive value of commonly used animal models, since some mechanistic approaches (eg, Substance P antagonists) that demonstrated efficacy in animal models did not demonstrate efficacy in human chronic pain conditions. There are several possible reasons that might explain the challenges in translating preclinical to clinical outcomes including pharmacokinetics, clinical study design, numbers of subjects required to see an effect, lack of alignment between clinical versus preclinical outcome measures, perhaps inherent differences in the biology of nociception in rodent and man, and insufficient time to integrate the outcomes during the decade-long discovery/development process. Despite these limitations, current drug discovery strategies continue to rely on preclinical models as an influential role in advancement of novel therapeutic candidate candidates into clinical trials.

Horizons for Molecular Imaging of Pain: Uncharted Territory The following section highlights examples of how emerging imaging technologies might be applied to pain. Many are not yet translatable to humans, and further experiments are required to determine the suitability of applying such new methodologies to the neuroimaging of pain.

Optical Imaging Fluorescence-mediated molecular tomography (FMT) is a technique that can 3-dimensionally image gene expression by resolving fluorescence activation in deep tissues. Application of near infrared fluorescent probes allows for deeper propagation of signals into tissues as compared with traditional fluorochromes and holds promise for deep tissue imaging.86 Bioluminescent imaging combines molecular genetics with optical approaches to allow visualization of fluorescent or biophotonic signals in living tissues. Transgenic mice that express genes tagged with fluorescent or bioluminescent molecules can be visualized noninvasively for dynamic monitoring of changes in gene expression in vivo.136 This approach has allowed noninvasive detection of immediate early gene expression in brain.5 The application of c-fos mice to investigations of pain would allow visualization of neuronal activity in vivo in preclinical models, perhaps illuminating neuroplastic mechanisms associated with pain and effects of targeted therapies on gene expression dynamically. Likewise, Arc GFP mice122 would also represent a suitable model particularly for investigating neuroplasticity (see below).

Stephenson and Arneric Biophotonic imaging has been applied to delineate markers of specific CNS cell types in vivo. Glial fibrillary acidic protein (GFAP) luciferase mice can be imaged to dynamically monitor astrocyte response to neuronal cell death in vivo.141 Given that GFAP activation occurs in models of chronic pain44 and that astrocytes have been implicated in the spread of pain via gap junction mediated astrocyte communication,49,127 application of GFAP transgenic mice to investigating pain will be a potentially powerful approach to investigate the effects of therapeutic agents in vivo, in particular, for therapies targeted at modulating neuroinflammatory processes.

Multiphoton Microscopy Multiphoton microscopy (MPM) is a method that allows in vivo visualization of fluorescence to image subcellular events using relatively deep optical penetration.128 Neuroscientists have benefited tremendously from this technology; an optical window at the surface of the brain allows direct cellular imaging of the brain in vivo. CNS applications to date include imaging of calcium, sodium, neuronal dynamics, and Alzheimer’s plaque pathology. Direct visualization of reactive oxygen species (ROS) in vivo has been shown in the brain of mice in a mouse model of AD by using multiphoton microscopy. Amplex red is a fluorogenic oxidation reporter agent and well-established reporter of free radicals.106 Visualization of Amplex red in the brains of transgenic amyloid depositing mice highlighted that ROS were observed in the regions of pathology.77 Furthermore, the same investigators demonstrated that antioxidant treatment diminished the levels of oxidative stress in the area of pathology.41 Reactive oxygen species, including nitric oxide,65 superoxide,119 nitrotyrosine, and peroxynitrite,67 have also been implicated in neuropathic pain. Application of MPM techniques to the spinal cord of rodent models of pain might be a suitable method to demonstrate the presence of ROS mediators in injured spinal cord as well as evaluate effects of putative therapeutic agents.

Important Approaches for Future Pain Translational Development The content presented in this next section represents advances that, to date, have little to no clinical imaging support. Emerging exploratory topics are reviewed in context with new imaging tools and technologies. It is hoped that this information will lead to new ideas and opportunities for exploratory research and technology development in the area of pain.

Functional Molecular Imaging of Cellular Dynamics Neuroplasticity In patients with neuropathic pain, fMRI has shown that during allodynic stimulation, there is recruitment of additional brain activations compared with reference pain network. Post-lesional shifts in activity between brain

573 hemispheres have been described by using neuroimaging approaches in a variety of syndromes such as poststroke brain reorganization98 and are interpreted as representing cerebral plasticity. Additional evidence of plasticity in neuropathic pain syndromes is supported by individual case reports in which pain states are modulated by selected brain lesions.26,88 Neuroimaging modalities that are specifically suited to imaging neuroplasticity include imaging glucose metabolism (FDG-PET) and imaging blood flow (fMRI). In preclinical applications, the application of optical imaging of immediate early gene mice (Arc, c-fos, described above, optical imaging) as well as other optical imaging approaches such as optical coherence tomography (OCT) are of relevance. A recent study described the application of OCT in mice to image neuronal plasticity associated with response to peripheral neuropathic pain.89 OCT provides high spatial resolution for cross-sectional imaging of living tissue and measures the intensity of infrared light backscattered from within target tissue. Increases in scattering intensity of near-infrared light were observed by OCT in cortical regions that are consistent with those showing increases in neuronal activity after peripheral nerve injury. Imaging neuroplasticity in transgenic bioluminescent mice is another approach to consider. Several different transgenic constructs have been developed where activity related genes can be visualized in vivo. Transgenic mice highlighting c-fos gene expression,5 postsynaptic density proteins (PSDs) tagged with GFP,34 and Arc GFP transgenic mice122 may be viable models for visualizing neuroplasticity of pain by subjecting the mice to conditions that elicit acute or chronic pain states. The application of c-fos mice to investigations of pain would allow visualization of neuronal activity in vivo in preclinical models, perhaps illuminating neuroplastic mechanisms associated with pain and effects of targeted therapies on gene expression dynamically. Because modulation of neuroplasticity has therapeutic implications for the treatment of neuropathic pain,24 imaging neuroplasticity holds promise for discovery and evaluation of new therapies.

Neurogenesis Hippocampal neurogenesis has been implicated in learning and memory and has been strongly linked to depression and stress.94 Clinical observations demonstrate that chronic pain subjects often have depression, experience chronic stress, and may experience cognitive dysfunction. Indeed, preclinical studies in rats subjected to acute and chronic pain stimuli have demonstrated reduced neurogenesis in the hippocampus as well as stressrelated changes in gene expression under conditions of persistent inflammatory pain.33 Very recent evidence suggests that it is possible to identify neural stem/progenitor cells in human brain by proton MRS imaging.73 It has been suggested that an important affective or emotional component of pain may affect cognition. Given the significant advances have been made recently in assessment of neurogenesis, imaging of modalities associated with stress and depression may provide a secondary

574 index of pain associated syndromes that are functionally linked to quality of life.

Apoptosis Imaging of apoptosis has been shown using targeted contrast detection by MRI,140 allowing in vivo imaging of tumors in rodents. Imaging of apoptosis has also been demonstrated by SPECT. Using a radiolabeled 99mTc-labeled annexin V probe, Blankenberg et al8 visualized hepatic apoptosis in a mouse model of hepatitis. There is substantial evidence that apoptosis takes place in the spinal cord under conditions of neuropathic pain.71,125 Imaging of apoptosis in animal models of pain would be an interesting yet technically challenging endeavor. Challenges include the site of injury (ie, the spinal cord is not easy to image even using conventional imaging approaches) and that there are a relatively small number of cells that undergo apoptosis in pain models (it is not yet known the number of cells that need to be undergoing apoptosis required for visualization). Future technologic advances probably will be required before such approaches are feasible.

Nanoimaging Technology MRI in conjunction with targeted imaging agents has been successfully applied to visualization of specific molecular events. This approach allows ultrasensitive imaging of specific biologic targets as opposed to assessing the overall structure of specific target organs. In the field of oncology, nanoparticle imaging has been successful in diagnosis, monitoring of progression, as well as targeting of specific therapies.15 New magnetic nanoprobes are being developed that can be conjugated with antibodies and show enhanced MRI sensitivity for detecting smaller structures.62 As detailed below, such innovative and emerging approaches hold tremendous promise for pain research.

Transcriptional Factors Imaging of reporter gene activity has yielded a promising advance in the ability to assess in vivo functional activation of inflammatory mediators. The transcription factor NF-␬B is ubiquitously expressed in virtually all cells and plays a role in proliferation, cellular activation, and apoptosis. Inappropriate regulation of the transcription factor NF-␬B has been implicated in a variety of human inflammatory conditions including cancer, arthritis, cardiovascular disease, stroke, AD, asthma, and inflammatory bowel disease.35 NF-␬B activation turns on a whole variety of important proinflammatory mediators; thus, modulation of transcription factor activity can have profound consequences at attenuating a multitude of downstream mediators. In vivo imaging methods have been developed in transgenic mice that permit visualization of functional NF-␬B activation in living animals.17 Classic inducers of NF-␬B activation reportedly upregulate target luciferase gene expression in target tissues; furthermore, NF-␬B activity reportedly is increased in joints in a rodent model of rheumatoid arthritis.17 An

Neuroimaging of Pain: Advances and Future Prospects important therapeutic molecular target in the NF-␬B cascade is an inhibitory subunit, IKK, which is composed of at least 2 subunits.139 Transgenic mice in which the luciferase is tagged with the IKK-␣ subunit have been developed and shown to be suited to imaging pharmacologic modulation of IKK activation in vivo.46 NF-␬B activation occurs in regions of pathology in multiple models of pain. In rodent models of pain, attenuation of NF-␬B activity directly or by modulation of IKK activity has beneficial effects on pain responses.55,111 Imaging NF-␬B in pain models promises to illuminate tissues in which functional activation of NF-␬B occurs as well as evaluating specific therapeutic agents targeting the NF-␬B signaling pathway.

Microglia In vivo imaging of microglia is possible in both humans and animals by using a selective PET radiotracer (PK-11195). PK-11195 selectively labels the peripheral benzodiazepine receptor (PBR), a protein that is selectively upregulated on activated microglia.3 Application of PK-11195 to a variety of human CNS disorders as well as animal models of CNS injury100,107 demonstrates that microglia specifically express PBRs after CNS injury. Using PET neuroimaging with PK-11195, microglial activation can be followed temporally in vivo.3 Given the wealth of recent information that implicates the critical role of microglia in mediating chronic pain states such as neuropathic pain,48,84,117,123 noninvasive detection of microglial signaling holds the promise of representing a tool for the diagnosis and management of neuropathic pain. Technical challenges in applying this in the near future include poor signal-to-noise ratio for this radiotracer as well as difficulty of imaging the spinal cord. Alternative microglia targeted imaging agents may hold promise for overcoming some of the technical challenges.56

Immune Cells The contribution of immune and inflammatory systems to neuropathic pain has been a topic of much investigation.79 Infiltration of inflammatory cells and breakdown of the blood spinal barrier occur in animal models of chronic pain.52 In addition, pain is a common symptom in patients with immune-mediated demyelinating neuropathies such as multiple sclerosis.81 In vivo imaging of leukocyte trafficking has been described by using intravital microscopy.78 Dynamic imaging of T-cell development has been studied in lymph nodes using 2-photon laser scanning microscopy.13 In vivo monitoring of macrophage migration into sites of injury after nerve injury has been described by using MRI in combination with administration of superparamagnetic iron oxide particles.7 It is important to understand the limit of sensitivity and resolution for these immune-mediated imaging approaches, given that the magnitude and extent of inflammatory pathology in neuropathic pain is not particularly profound.

Stephenson and Arneric

Prospects in Drug Development: Targeting Diverse Patient Populations With Diverse Pain States Genetic factors contribute to pain susceptibility. It is well known that there are interindividual differences in pain perception, and some pain disorders are known to have genetic origin, for example, familial hemiplegic migraine. Association studies have implicated that several candidate genes may be associated with pain perception.103 Mutations in the gene encoding the sodium channel Nav1.7 can result in an inability to perceive pain25; alternatively, a gain of function mutations in the same gene results in enhanced pain perception; the result is an autosomal dominant disorder, erythromelalgia, in which patients experience burning pain in response to warm stimuli or modest exercise.31 These fascinating results suggest that the Nav1.7 gene is a target for developing new therapies to modulate pain processes. Imaging of patients with these disorders would be a key area of pain research directly linking genetics to both loss and gain of human nociceptive function. An example of correlation between pain and genetic susceptibility was described by Zubieta et al.142 Individuals homozygous for a polymorphism in the COMT gene showed higher pain ratings as well as reduced ␮-opioid system responses to pain as assessed by PET.142 Neuroimaging of subjects with particular genetic susceptibilities may be able to help select patients that will benefit or be refractory to novel therapies. In this way, molecular targeting of patients will be possible, as has recently become possible with breast cancer treatment.108 Improving our understanding of the CNS mechanisms that underlie pain processing may yield new targets for treatment of the behavioral-cognitive and pharmacologic aspects of pain conditions. Different types of pain syndromes (acute pain, experimental hyperalgesia, and chronic pain) have expectedly shown different responses in brain.68 Thus, defining the type of pain state could be

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575 a very important application of neuroimaging to define the patient populations that would be amenable to specific therapeutic interventions. In this way, functional neuroimaging could play a role as a diagnostic/evaluative tool in chronic pain. More importantly is the potential for applying neuroimaging to evaluation of pharmacologic treatment approaches. Using imaging to assess treatments that are neurorestorative may provide novel avenues to treat chronic pain. Objective measures of responders versus nonresponders as well as defining specific patient populations that can benefit from targeted therapies have tremendous potential.11 Application of imaging in clinical trials promises to help with diagnosis of a patient’s pain condition in a more objective and robust way, further enabling more effective targeting of therapies to effectively treat chronic pain conditions. In summary, chronic pain represents a seriously debilitating and complex process in need of new and effective therapies. Application of neuroimaging to noninvasive assessment of pain has elucidated anatomic and functional processes previously unknown. Innovations in new emerging molecular imaging technologies hold tremendous promise for diagnosis, management, and improved treatment for patients who have pain. In preclinical models, application of imaging represents a method for identification of new translational tools that probably will improve the predictive value and accurate advancement of many promising new therapies. Improvements in therapy that may benefit from increased attention to neuroimaging of pain include better clinical risk stratification, more optimal selection of patient populations amenable to specific disease interventions, and improved assessment of treatment efficacy.

Acknowledgments Gratitude is extended to Dr. Paul Maguire, PGRD, Global Clinical Technologies, and Dr. Michael Rigby, Discovery Biology, PGRD, for their careful review of the manuscript.

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