Molecular Mechanism of Pain

CHAPTER 10

Molecular Mechanisms of Pain: A Basis for Chronic Pain andTherapeutic Approaches Based on the Cell and the Gene

Jonathan Riley, B.S.E., and Nicholas M. Boulis, M.D.

INTRODUCTION

The perception of pain serves the adaptive role of provid-ing an affective context for real, impending, or perceived

tissue damage. In order to accomplish this critical role,separate classes of peripheral receptors serve to characterizeand relay the presence of noxious stimuli. The initial stimuliis processed and filtered in a stepwise fashion in the periph-ery, throughout the neuraxis, and in the telencephalon. Im-portant molecular events contribute to signal processing andmodulation within the periphery, dorsal root ganglion, spinalcord, and brainstem, as well as in specific subcortical nucleiand cortical pathways.

In order for pain to act as an effective motivator,systems must exist to identify the source and location of thepain, as well as to attach a powerful emotional valence to theperception. Even when the direct insult, resulting in a “firstpain” has passed, the nociceptive sensation can be prolongedby inflammatory mediators that are released into the sur-rounding milieu. The result is a more diffuse “second pain,”characterized by a burning quality, which plays the physio-logical role of helping to prevent further insult while ahealing response occurs. In addition to physiological noci-ception, which produces sensations in direct or indirect re-sponse to noxious stimuli, a pathological form, neuropathicpain, results from an underlying dysfunction in the nervoussystem. Often, neuropathic pain is the result of physicaldamage to a nerve. In fact, animal models of neuropathic painfocus largely on compression or partial transection. However,neuropathic pain can be temporally and spatially abstractedfrom the inciting damage, as in the case of phantom limbpain. This abstraction results from neuroplasticity in theperiphery and within the central nervous system. These formsof plasticity are known respectively as peripheral sensitiza-tion and central sensitization. Through these processes, painperception shifts out of proportion with the magnitude ofafferent input from the periphery resulting in hyperalgesia orallodynia. Chronic neuropathic pain results when the percep-tion becomes entirely independent of stimulation.

Because the painful sensation has been at least partiallyabstracted from the stimuli as a consequence of centralplasticity, and because rostral transmission does not seem tobe confined to a single fiber tract, neuropathic pain hasproven difficult to treat by conventional pharmacological andsurgical techniques. Current strategies have been, at best,incompletely successful because they fail to fully account forboth the unique anatomic and pharmacological implicationsof this dynamic, pathological process. Within a given locationof the nociceptive processing network, multiple molecularevents, including multiple receptors and ligands, encode pain.Thus, surgical therapies that possess only anatomic specific-ity, inherently lack pharmacological specificity. In contrast,analgesic drugs can target specific neurotransmitter systems,but lack anatomic specificity. The body uses individual trans-mitter/receptor systems in multiple physiological roles. Forthis reason, design of more ideal treatments will requireachievement of both forms of specificity, to reverse or maskthe processes underlying neuropathic pain while allowingretention of the capacity for nociceptive transmission. Assuch, interest has developed in novel treatments for neuro-pathic pain that locally manipulate signal transmission at amolecular and cellular level.

This chapter will explore cellular and genetic therapeu-tic mechanisms designed to reverse or mask neuropathic pain.To provide a context for the discussion of the molecularevents that underlie pain, the anatomical and pharmacologicalbasis for nociception and chronic pain will follow. A discus-sion of nociceptive pain requires an introduction to thefunctional anatomy with an emphasis placed upon the mo-lecular basis of pain transmission and theorized methods ofregulation. An understanding of the pain transmission appa-ratus, at macroscopic and molecular levels, will provide thebackground necessary to discuss aspects of peripheral andcentral sensitization which presage, in pathologic conditions,the development of neuropathic pain. Once the basis for thecurrent understanding of neuropathic pain has been summa-rized, an exploration of the merits and limitations of currentpharmacologic techniques will help to illustrate the need formore advanced treatments.

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The development of advanced cell and gene basedtherapeutic techniques will be introduced as a potential meansof circumventing these limitations by providing a platformthrough which anatomic and pharmacological specificity maysimultaneously be achieved. An emphasis will be placed onpotential future avenues of research.

FUNCTIONAL ANATOMY OF PAINTRANSMISSION

Peripheral Stimuli Detection andTransmission2,3,5,19,22

Sensation of noxious stimuli requires that the incomingstimuli be converted into a form that can be interpreted by theneural networks of the peripheral nervous system (PNS) andcentral nervous system (CNS). Afferent fibers are able topreserve the information content of incoming stimuli by usingsomatotopic fiber organization in tandem with the ability toconvert analog afferents into frequency-modulated signals.This provides the ability to convey the location of sensation,the magnitude of stimulation, and the duration of occurrence.In the specialized case of nociception, mechanisms are re-quired to detect temperature, chemicals, and applied force.Nociception is encoded by receptors that are primarily con-cerned with these other modalities through an activationthreshold. That is, pain occurs when mechanical, chemical,and thermal receptor are over-activated (Table 10.1).

One of the important detectors of noxious stimuli is thetransient receptor potential (TRP) channel. The family ofTRP channels is characterized by homology. Each of the TRPchannels has six transmembrane subunits, and various sub-families are known to have disparate functions in differentorgan systems and across species. With respect to the detec-tion of noxious stimuli, the TRP channels known to be ofinterest include TRPV1–4, TRPM8, and TRPA1. Each chan-nel is unique, responding to different stimuli. TRPV1–4 allrespond to heat and are Ca2� permeable, though to differentdegrees. Each has a unique threshold for temperature detec-tion. The minimum thresholds for activation have been re-spectively reported as 43oC, 52oC, 31oC, and 25oC. In con-trast, TRPM8 and TRPA1 are cold-sensing receptors with

maximum thermal detection thresholds of approximately25oC and 18oC, respectively.

Thus far, evidence has only linked these specific TRPchannels to noxious thermal stimuli in the periphery. Theo-ries for the mechanism of signal transduction are least welldeveloped for encoding thermal energy, when compared withsome of the other known sensory roles in which TRP chan-nels function. Some hypotheses include a temperature-depen-dent change in membrane surface tension, reversible channeldenaturation, and cytoplasmic binding of diffusible secondmessengers. All three of these hypotheses rely upon revers-ible plasticity of the channel conformation to allow for eithera direct or indirect increase in ion flux.

In addition to the TRP channel providing a mechanismfor thermal energy transduction, the DEG, DRASIC, andTREK-1 channels are mechanosensors involved with detec-tion of noxious, stimuli. The P2X3, DRASIC, ASIC, andTRPV1 channels are chemoreceptors also known to contrib-ute to nociception. These receptors are categorized in Table10.1.

Peripheral excitability represents a continuum whichcan be altered in response to exceedingly noxious stimuli, orunder conditions present after damage to neural structures. Inlight of this, it is important to keep in mind that the sensoryreceptors are responsible for a graded depolarization withindendrites. The excitability of the relay neuron depends on thecomplement of ion channels responsible for re-encodingincoming analog signals as action potentials, for afferenttransmission. These neurons, located within the dorsal rootganglion (DRG), contain a variety of Na�, Ca2�, and K� ionchannels, the presence of which can be modulated dependingupon several factors, including the duration and magnitude ofnoxious stimuli applied to the periphery. Briefly, the Na�

channels are dichotomously considered as being tetrodotoxin(TTX) sensitive or resistant. The role of these channels andthe effect of their modulation on pain perception will bereviewed in the later discussion on peripheral sensitization.

Signals generated from the peripheral nociceptors arecarried by afferent fibers to specific laminae of the spinal corddorsal horn. Such afferent nociceptive transmission is thoughtto occur via a combination of thinly myelinated A� fibers andunmyelinated C fibers. The former have conduction velocitieson the order of 5–30m/s while the latter transmit at less than1m/s. Both thermal and mechanical nociceptive fibers use A�fibers while thin, unmyelinated C fibers provide a conduit forpolymodal nociception, which responds to noxious chemical,mechanical, and thermal stimuli. Under physiologic condi-tions these nociceptors require high threshold stimuli toinduce action potential generation. However, peripheral sen-sitization can lower the threshold to incite nociceptive stimulitransmission, hence allowing trivial stimuli to trigger pain.

TABLE 10.1

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CENTRAL TRANSMISSION ANDREGULATION2,3,32,42,43

Primary sensory (DRG) neurons synapse within specificlaminae of the dorsal horn as shown in Figure 10.1. The graymatter of the spinal cord is traditionally subdivided into 10specific laminae based on cytoarchitectural criteria; the dorsalhorn contains six of these subdivisions. The predominant sen-sory layers, beginning dorsally, are: marginal (I), substantiagelatinosa (II), nucleus proprius (III-IV), and deep layers (V–VI). The majority of general sensory afferents synapse in Lam-ina V, with significant contributions also being made to LaminaeIII and IV. The marginal layer is almost exclusively comprisedof input from nociceptive afferents that project to higher regu-latory centers, including both A� and C fibers. A� fibers alsosynapse in Lamina V onto wide dynamic rage neurons (WDR),whereas the other primary contribution of C fibers is in LaminaII. A generalized distinction exists between the C fibers inner-vating Lamina I and the outer portion of Lamina II and those Cfibers innervating the inner portion of Lamina II. The formerrespond more to heat and chemicals, contain neuropeptides, suchas Substance P (SP) and calcitonin gene-related peptide(CGRP), and tend to synapse on second order neurons projectingto higher centers. The latter contain a smaller complement ofneuropeptides and synapse on interneurons in Lamina II. Lam-ina II is largely composed of interneurons of both excitatory andinhibitory function. Lamina III to IV receives largely A� fiberstransmitting non-noxious stimuli while maintaining high spatialresolution. Lamina V, predominantly receive A� wide dynamic

range (WDR) input and A� input. Additionally, WDR neuronsare capable of receiving input directly from C fibers, as theirdendrites may extend dorsally to superficial laminae, or indi-rectly, due to the contribution of interneurons.

Glutamate neurotransmission underlies much of pe-ripheral pain transmission. It typically functions throughnon-NMDA (AMPA kainate) and metabotropic (mGluR)receptors under conditions of low frequency, high thresholdstimulation. In contrast, the NMDA glutamate receptor(NMDAR) seems to be a strong contributor to the process ofcentral sensitization. The NMDAR plays a minor role inmembrane depolarization under normal circumstances. Theprimary balance of intracellular phosphatase-kinase activityprevents the receptor from being activated via phosphoryla-tion, except in the presence of mediators promoting a state ofsensitization. Additionally, at the resting membrane potential,a Mg2� plug is present, preventing ion flux. This process isdepicted in Figure 10.2.

Primary inhibitory neurotransmitters acting at the levelof the dorsal horn include GABA and glycine. They haveboth pre- and postsynaptic functions with the majority actingthrough postsynaptic mechanisms. Both neurotransmittersactivate Cl- ion channels, through GABAA and specific gly-cine channels, respectively. GABA is also known to actpostsynaptically through the GABAB metabotropic ion chan-nel, which ultimately achieves the same hyperpolarizingeffect through differing modulation of K�, Ca�, & Na�

channels.

FIGURE 10.1 Rexed’s lamina. Input and output synapses usepredominantly excitatory amino acid transmission (Gluta-mate). Inhibitory interneurons (GABA and Glycine) play a rolein modulation of the nociceptive signal, as do endogenousopiates. Fibers descending from the brainstem further regulatenociceptive signal transduction through catecholamine trans-mitters.

FIGURE 10.2 NMDAR activation in central sensitization. A,under normal circumstances, afferent glutamatergic transmis-sion is handled by multiple channel types (e.g., AMPA, kainate,mGluR). Descending modulation seems to be primarily inhib-itory and mediated by GABA and glycine under normal con-ditions. B, enhanced afferent signaling to the dorsal horn, inconjunction with changes in descending modulatory tone canpromote central wind-up. C, after NMDAR activation, secondorder afferent signalling may be abstracted from peripheralinput.

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Central sensitization depends on a change in the bal-ance between excitatory and inhibitory transmission withinRexed’s lamina. This balance is largely achieved as a resultof the post-translational and transcriptional mechanisms thatmodify the activity and quantity of these receptors. Theultimate result of these changes in activity and expression isan increase in excitability of the afferent tracts responsible forpain transduction. This increased excitability uncouples pe-ripheral receptor activity from the affective sensory experi-ences of pain.

ASCENDING PATHWAYS2,43,49

A minimum of five pathways have been identified asproviding conduits for afferent nociceptive neurons. Contri-butions are provided by the spinothalamic, spinomesence-phalic, spinoreticular, spinohypothalamic, and cervicotha-lamic tracts. These pathways track through eitheranterolateral or dorsal fasciculi.

The Anterolateral QuadrantThe neospinothalamic tract, or ventral spinothalamic

tract, advances to the ventral posterior lateral (VPL) nucleusof the thalamus (alternatively termed ventralis caudalis [Vc]),and is implicated in the discriminatory sensation of pain.Fibers from the neospinothalamic tract are primarily WDRneurons, projecting from deeper dorsal horn laminae (IV–VI).The paleospinothalamic, or dorsal spinothalamic, tracts isimplicated in the affective aspect of pain. These tracts arepredominately nociceptive neurons projecting from moresuperficial laminae (I–II). Terminations are largely in medialportions of the thalamus. Spinoreticular neurons, largelyoriginating from Laminae VII and VIII of the dorsal horn,terminate within the brainstem. Axons passing to the medullahave been shown to terminate in nucleus retroambiguus,superspinalis, and the dorsal and ventral aspects of the me-dullae oblongatae centralis. Advancing rostrally, terminationsoccur in the lateral reticular nuclei, the nucleus gigantocellu-laris, paragigantocellularis dorsalis and lateralis, and the nu-clei pontis caudalis and oralis. This tract affects autonomicregulation and awareness, through the reticular formation andprojections to the hypothalamus and thalamus. Contributionis also thought to be made to a “spino-limbic” tract throughmultisynaptic connections from the midbrain to the hypothal-amus, medial thalamus, and limbic system.

The majority of afferents of the spinomesencephalictract originate in Laminae I and IV to VI. Although collater-als to the lateral thalamus exist, the majority of the fibers passto midbrain nuclei. The afferent fibers exhibit rough somato-topy, with afferents from more inferior fibers terminatingcaudally within the midbrain. Terminations include the peri-aqueductal gray matter (PAG), anterior and posterior pretec-tal nuclei, and nucleus cuneiformis. The PAG is an important

modulator of the spinal mechanisms of descending analgesicmodulation.

The Dorsolateral QuadrantA spinocervicothalamic pathway has been noted in

multiple species, arising and passing through a contralateralposition at the cervical levels C1 and C2. Animal studies haveindicated termination in the lateral cervical nucleus, whichprojects to the lateral thalamic nuclei. This pathway as aconduit for nociception.

Evidence exists to support the conduction of visceralnociceptive activity within tracts of the dorsal column. First,extreme pain is triggered during mechanical probing of thedorsal fasciculus. Second, partial midline myelotomy hasbeen successfully used to treat pelvic cancer pain.

CORTICAL PATHWAYS AND AFFECTIVEATTACHMENT13,36,40

Nociceptive projections to the brainstem and deep brainnuclei either activate descending regulatory mechanisms orrelay to cortical structures that mediate our ability to localizepain and motivate protective behavior. This latter activityrequires pain to elicit negative reinforcement, which occursthrough the limbic structures underlying emotion.

The initial transmission of afferents into subcorticalnuclei and to cortical matter seems to contribute to parallelpathways, each constructed of serial connections, which ul-timately integrate. Afferents to brainstem structures, such asthe reticular formation, largely from the spinoreticular tract,and to subcortical structures, such as the hypothalamus andamygdala, are capable of generating immediate autonomicresponses, resulting in arousal, modulation of autonomic toneand sympathetic output, and providing an appropriate emo-tional context.

Spinothalamic fibers terminate in the thalamus withlarge contributions to the lateral thalamus (VPL, VPM, VPI)as well as to the medial thalamus (CL, CM, Pf, and VMpo).Projections to the lateral thalamus continue largely to the S1

somatosensory cortex. Tracts originating in the VPI project toS2. Fibers terminating in the medial thalamus provide a non-or loosely somatotopic bilateral representation. Medial tha-lamic fibers from the VMpo pass towards the insular cortex.Thus, lateral thalamic and VMpo serve to spatially andtemporally localize pain.

It has been postulated that nociceptive fibers form along serial tract, coursing from S1 through parietal associa-tion areas of S2, to the insular cortex, and ultimately to limbicstructures, including the amygdala and hippocampus. Thisprovides a more integrated view in which the discriminativeaspect of pain is merged with the affective component byprojections between the VPL-S1–2-IC circuit and the limbicsystem. This converges with direct spinal inputs to the amyg-dala. A mechanism for marrying the discriminative-affective



components of pain with executive and motivational func-tions is provided via connections of the insular cortex withthe anterior cingulate cortex (ACC). Putatively, this connec-tion merges the ability of the aforementioned serial circuit tomaintain integrity of discriminative data regarding the loca-tion and magnitude of the stimuli in relation to other, poten-tially concomitant, stimuli with executive and motivationalcapabilities, through additional ACC connectivity.

Imaging studies show the ACC to be one of the mostconsistently active regions in response to nociceptive stimuli,with connectivity to the prefrontal cortex and to premotorareas. Reciprocal projections between the ACC and prefron-tal cortex provide a mechanism for additional affective at-tachment, and for consolidation of decisions regarding avoid-ance behavior. Those tracts to the premotor cortex couldprovide the motivational stimuli necessary to drive the ap-propriate motor response. Additional loops through the pre-frontal cortex and limbic system could help to attach long-term affective significance to the inciting event. Thus,affective attachments are integrated with somatosensory lo-calization to allow prefrontal cortex to initiate avoidancebehavior (Fig. 10.3).

MECHANISMS OF SENSITIZATIONThe physiological process of nociception typically con-

sists of the sensation of first and second pains. The former iscomprised of a relatively distinct sensation (e.g., sharp, sim-ilar to a pin prick) localized to the area of the noxious insult.It is propagated largely via the A� fibers. In close temporalapproximation to the initial sensation, the second pain is amore diffusely localized sensation with less distinct bordersand a more general quality (e.g., dull and burning). Addition-ally, the second pain outlasts the sensation of the first pain.First pain provides an acute awareness of the presence andlocation of a noxious stimulant, motivating immediate escape

behavior to limit tissue damage. The process of tissue damageprovides a context for the development of a second pain.Sustained second pain (peripheral sensitization) depends onthe release of multiple mediators due to injury-related cyto-toxicity and the downstream effects thereof. The direct andindirect actions of these inflammatory mediators modulate analteration in membrane excitability of peripheral nerves,particularly the polymodal C fibers, contributing to a lasting,diffuse, and painful sensation. Whereas first pain is largelyinvolved in escape, second pain creates a stimulus for learn-ing and hence avoidance of future tissue damage.

PERIPHERAL SENSITIZATION7,37

Cytotoxicity, presumably due to a noxious insult,results in the release of inflammatory mediators into thesurrounding extracellular milieu. A list of mediators con-tributing to this process is provided in (Table 10.2). Theprocess of peripheral sensitization contains several posi-tive feedback loops, whereby nerves respond to localinflammatory mediators by releasing other molecules thatserve to further increase peripheral excitability. Prosta-glandins, produced by Phospholipase A2 from membranebound arachidonic acid, can act to sensitize nociceptorsand increase vascular permeability, along with many of theother mediators present. Orthodromic and antidromictransmission within the sensitized fibers can result in therelease of the neuropeptide SP at other peripheral endings.SP contributes to the positive feedback cycle through twoactions. In one, the presence of SP results in the release ofbradykinin from nearby vasculature. The downstream ef-fect of bradykinin results in an increase in vascular per-meability and helps to sensitize nociceptors. SP also actsdirectly on mast cells to induce histamine release and onplatelets to cause serotonin release, the presence of whichis aided by the effects of bradykinin on vascular perme-

FIGURE 10.3 Descending modulation, largely through the PAG, helps to govern rostral nociceptive transmission. Projections tothe medial thalamus continue to the insular cortex updating general bodily awareness. Somatotopy of afferent nociceptive signalsis preserved through fibers projecting to S1, with somatotopy preservation and initial abstraction potentially occurring in S2.Projections through the insular cortex to the limbic system are thought to be responsible for attaching an affective componentto the nociceptive stimuli. Convergence of these signals on the ACC and passage through the prefrontal cortex allows evaluationof the stimuli in context of the affective component attached to the stimuli and location of the insult. As a result, a consciousresponse to the nociceptive stimuli may be initiated



ability. These actions of SP and those of other mediatorsserve to utilize the induced inflammatory response tospread the activated receptive field and, thus, to increasenon-localized peripheral excitability. In large part, thisspread relies on the elevation of vascular permeabilitywhich sensitizes neuronal afferents and recruits additionalfibers through the influx and subsequent effects of inflam-matory mediators. Increased peripheral afferent excitabil-ity is accomplished via the effects of these mediators onposttranslational and transcriptional processes resulting inchanges to the proteins underlying peripheral nerve acti-vation. These changes affect sensitization along a contin-uum in physiologic, neurogenic, or neuropathic processes.This process is illustrated in Figure 10.4.

Molecular Mechanisms of PeripheralSensitization

Peripheral sensitization is ultimately mediated by theeffects that these inflammatory mediators exert at the molec-ular level. As excitability is determined by changes in ion fluxwhich modulate the transmembrane voltage, modulation oc-curs by two primary mechanisms. Ion flux can either bealtered in the receptors directly responding to noxious stimulior in the voltage-gated ion channels that determine parame-ters such as conduction velocity, activation threshold, andfiring inactivation. Proposed mechanisms of altered excitabil-ity involve either increased availability or increased activa-tion of these two types of channels.

Post-translational alteration of excitability occurs eitherthrough direct modulation of the receptor by allosteric inter-actions or through intracellular signaling pathways. An ex-ample of the former is thought to occur at the TRPV1receptor, in which the presence of either H� or capsaicin isthought to contribute directly to a reduction in the thermalactivation threshold. Increased neuronal excitability followsthe activation of cytoplasmic signaling cascades triggered viainflammatory mediators. Conserved signaling pathways uti-

lize PKA, PKC, MAPK, PLC, and other intermediaries, oftenin parallel, to modulate receptor function in response to thepresence of extracellular stimuli. Some receptors known to becoupled to PKA through Gs include the B1 receptor (brady-kinin), the EP2 (PGE2), and 5HT-1A (serotonin). Down-stream effects of PKA occur on tetrodotoxin sensitive andresistant (NaV1.8 and NaV1.9) Na� channels, TRPV1, andspecific classifications of Ca2� channels, notably the N-typechannel (CaV2.2), and on particular K� channel subtypes.Though candidates are present, the particular K� channelsinvolved in modulation of excitability have yet to be defini-tively isolated. PKC is thought to have similar actions on thesame receptors mentioned. NaV1.8 and NaV1.9 modificationsrequire coactivation of the PKC and PKA cascades. Stimu-

TABLE 10.2

FIGURE 10.4 Mechanistic basis of peripheral sensitization.Upon damage to tissue stroma a series of neuroactive agentsare released. Some mediators act directly upon the nociceptiveafferents while others act indirectly, through promoting ex-travasation of inflammatory cells and mediators. A, severalmediators (e.g., H�, K�, Serotonin, Histamine, Prostaglandin,Bradykinin) are released from damaged tissue. Prolonged pres-ence of these mediators, most notably prostaglandin, aids innociceptive afferent sensitization as well as in extravasation ofinflammatory mediators and cells from adjacent endothelium.B, sensitized afferents release Substance P which has dual roles.One of these roles is to cause liberation of Bradykinin fromvascular endothelium. This facilitates a positive feedback forcontinued sensitization by inducing continued sensitization ofnociceptive afferents. C, additionally, SP liberated from theterminals of activated nociceptive afferents activates bothplatelets and mast cells, causing release of serotonin andhistamine, respectively. Both contribute to the process ofperipheral sensitization.



latory phosphorylation of the Na� channels is thought todirectly alter the effects of action potential generation. Stim-ulatory phosphorylation of Ca2� channels can modulate mul-tiple pathways. Increased intracellular Ca2� can affect furtherPKC activation and additionally activates CaMK. Both ofthese activities are known to increase transcriptional activity.Elevated Ca2� levels can also contribute to synaptic vesiclefusion and antidromic release of neuropeptides from theperiphery, contributing to the positive feedback involved inperipheral sensitization.

The aforementioned mechanisms provide quick re-sponse to tissue damage or peripheral neuronal injury with anincreased excitability potentially translating to hyperalgesiaand allodynia. This sensitization motivates the individual toprotect against further tissue damage. Transcriptional mech-anisms largely result in a sustained increase in the comple-ment of nociceptive receptors, providing a mechanism forprolonged peripheral excitability. Members of the MAPKfamily (ERK, p38, and JNK) act to modulate phosphorylationof the transcription factors CREB, c-Jun, and ATF-3. Thisaltered phosphorylation permits the factors to translocate intothe nucleus, upregulating the production of inflammatorymediators, membrane receptors, and ion channels.

A CURRENT THEORY OF CENTRALSENSITIZATION1,22,35,41,48

Peripheral sensitization acts as a complex process inwhich redundant inflammatory mediators act through a seriesof processes to both recruit and increase excitability inafferent fibers. Central sensitization seems to be a mechanis-tically simpler process, largely governed by summation ofhigh frequency afferent signals occurring at synapses withinthe dorsal horn. Prolonged postsynaptic depolarization resultsin recruitment of previously inactive membrane receptors thatfurther increase postsynaptic excitability, ultimately resultingin posttranslational modification and contributing to tran-scriptional upregulation. In concert with changes in descend-ing brainstem modulation, these processes hypersensitizerostral transmission of noxious stimuli to or uncouple trans-mission from stimulation by peripheral afferents. The follow-ing attributes have been suggested to describe the process ofcentral sensitization: lowered discharge threshold present incentral nociceptors with consequent increases in the potentialfor spontaneous and posthumous discharge, a broadening ofthe peripheral receptor field, and a depressed ability forcentral inhibitory pathways to counteract the sensitization.Ultimately, this rewiring process and potential for dissocia-tion of perception from peripheral sensation provides a func-tional underpinning for the development of neuropathic pain.

Molecular Mechanisms of Central SensitizationThe mainstay of nociceptive transmission through the

dorsal horn occurs through AMPA, kainate, and mGluR

receptors, which are activated by presynaptic glutamate re-lease. Also present, but largely inactive, is the NMDAR.Under conditions of low frequency activation and normaltransmembrane voltage, glutamate is not sufficient to result inactivation. The channel is held in check by a Mg2� plug thatoccludes the conducting channel at the resting membranepotential. Additionally, it is held in check through the pre-dominant dephosphorylation of cytoplasmic portions ofNMDAR subunits. Sustained depolarization seen in highfrequency afferent transmission secondary to peripheral sen-sitization allows for removal of the plug. Under such circum-stances, additional voltage-gated Ca2� channels may also beactivated. This leads to the potential for further activation ofnon-specific cation channels which rely on elevated, intracel-lular [Ca2�].

Post-translational modification of the NMDAR is me-diated by both a receptor bound tyrosine kinase (Src) and acounter-regulator, striatal-enriched phosphatase (STEP).Basal transmission rates favor the activity of STEP. Modu-lation of Src activation, and thus NMDAR activation, repre-sents a convergence point of several intracellular signalingpathways. Intracellular cascades which impinge upon Src areactivated by the presence of pro-inflammatory molecules.The process of NMDAR activation in response to prolongedstimulation, via membrane depolarization or through activa-tion of Src is termed “wind-up.” A schematic overview of thisprocess is provided in Figure 10.5. In addition to the in-creased activation of the NMDA receptor, evidence indicatesthat secondary neurons also become less responsive to inhib-itory stimuli, such as GABA and glycine, during sensitiza-tion. Recent data correlates activation of the NMDA channelwith the postsynaptic presence of superoxides. Putatively, theelevated influx of Ca2� aids in production of superoxides.Evidence has also been provided to indicate that the super-oxides react with NO, produced by the neuronal NOS variant,nNos, to produce peroxynitrites. These molecules function tobind and inactivate superoxide dismutase (SOD), in a processthat promotes hyperalgesia through downstream effects. Bothintroduction of SOD mimetics and inhibition of NO produc-tion have significantly attenuated this hyperalgesic effect.

Analogous to the mechanisms occurring within theperiphery, post-translational and transcriptional modulationoccur along a continuum. Kinases, notably one of the MAPKfamily members, ERK, are responsible for phosphorylatingsuch transcription factors as c-Fos, ELK-1, and CREB. Ad-ditionally, inflammatory cytokines contributed from the sur-rounding milieu, including spinal glia, contribute to activa-tion of the transcriptional processes which increases centralexcitability. Inflammatory mediators such as SP are upregu-lated in surrounding A� fibers, potentially resulting in allo-dynia. The presence of reactive oxygen species, mentionedearlier, can also trigger the translocation of some redoxsensitive transcription factors, such as NF-KB and AP-1,



which signal the production of inflammatory cytokines. Thus,while induction of central sensitization is primarily caused byfrequency-dependent depolarization, mediators from the sur-rounding environment appear to play a large role in aiding thetransition from a stimuli-dependent transient excitation to onethat persists, functioning independently of inciting stimuli.

CORTICAL AND SPINAL-BRAINSTEMMODULATION47

Regulatory modulation of the affect associated with apainful stimulus can occur at each of the levels listed. Intra-cortical mechanisms of pain modulation could functionlargely by dissociating an afferent nociceptive stimulus fromits affective component. Under situations of extreme physi-ological stress, this provides clear survival benefits. Suchmodulation likely explains the initially attenuated affect thatsoldiers and professional athletes sometimes attach to seriousinjury, as compared to others in less extreme circumstances.To complement the potential for intracortical modulation, alarge body of evidence suggests that both cortical and brain-stem structures course caudally to directly converge uponafferent nociceptive signals at the level of individual spinalsegments.

Determination of whether or not afferent signals will besent rostrally via second order neurons in response to periph-eral stimulation is thought to be governed by the principles ofHebbian signaling and gate control theory. Nociceptive pro-

jection neurons receive projections from first order nocicep-tive afferents, non-nociceptive afferents, descending modula-tory efferents, and inhibitory interneurons. Non-nociceptiveafferents serve to decrease afferent nociceptive signaling byincreasing tonic signaling through inhibitory interneurons.This is the biological underpinning of the analgesic use oftranscutaneous electrode nerve stimulator (TENS). A simplerapplication of the same principle is to rapidly shake one’shand following a nociceptive insult (e.g., a cut). Descendingmodulatory fibers can interact either pre- or postsynapticallyto modulate neurotransmitter release or excitability. In thisway, descending modulation provides an additional meansfor third party plasticity. Synaptic strength, then, is altered bymediators in the surrounding milieu, local inhibitory inter-neurons, the presence of non-noxious afferents, frequency oftransmission from the primary nociceptive afferent, and thepresence of descending modulatory fibers.

Basal Tone: Primary and Secondary NeuronalPools

Multiple modulatory brainstem nuclei have been dem-onstrated to contribute to descending modulation. Projectionsfrom the PAG to the rostroventral medulla (RVM) have beenparticularly well studied. Further, the RVM contains othernuclei that are of particular interest in descending modula-tion. Such nuclei include the nucleus raphe magnus (NRM),nucleus gigantocellularis pars �, and the nucleus paragigan-

FIGURE 10.5 Mechanisms of central sensitization. Both NMDAR and non-NMDAR postsynaptic receptors are responsive toGlutamate. A, under physiological conditions non-NMDAR glutamate receptors produce the depolarization necessary to createAPs for afferent signaling. NMDAR channels are not thought to conduct significant current under physiologic conductionfrequencies. B, high frequency stimulation is thought to remove the Mg2� plug from the NMDAR channel pore as an artifact ofelectrostatic repulsion mediated by prolonged membrane depolarization. Glycine binding is also required as a cofactor (notshown).C, once activated, a series of intracellular changes occurs to facilitate central “windup” by ensuring continued NMDARactivation and presence. These include both posttranslational and transcriptional events. The end result is that abstraction mayoccur; peripheral input may not be required, or may be required to a lessened degree, for afferent signaling (not shown).



tocellularis lateralis. Evidence has accumulated which indi-cates that PAG receives both cortical efferents and stimulifrom ascending tracts. Projections from the PAG to the RVMseem to provide indirect contribution to the descending mod-ulation of nociceptive afferents by coursing through thedorsolateral fasciculus. Work with rat models indicates thatunder physiological conditions, a net tonic signal seems to beinhibitory and at least partially governed by descendingserotonergic output from the NRM, as well as output from thelocus coeruleus. In addition to a tonic inhibitory output, themagnitude of inhibition was also shown to increase followingnoxious insult mimicking that of peripheral inflammation,seemingly as a mechanism to depress a central sensitization.To complement descending inhibition, dorsal horn neuronsbecome more attuned to this signaling, potentially due to anincrease of receptors or receptor activation in response toinhibition by opioids and noradrenergic stimulation.

Descending modulation of nociception can be facilita-tory as well as inhibitory. The majority of studies that suggestdescending mechanisms for facilitation refer to effects onsecondary neuronal pools. Antagonists for neurotensin andNMDAR have been shown to prevent secondary facilitation.Furthermore, these receptors have been shown to exist withinpathways from the PAG to the RVM. Additionally, antago-nists to NO synthesis in this pathway have prevented de-scending facilitation in animal models, indicating importantroles for the NMDAR, neurotensin receptor, and NO.

CHRONIC PAIN: THE NEED FOR TREATMENTThe preponderance of data suggests that neuropathic

pain results from aberrant nociceptive signals originatingfrom a damaged pain processing system. Chronic pain is adistinct entity caused by central sensitization secondary eitherto chronic peripheral tissue damage and inflammation, nervedamage, or a combination of both. The local environment andaltered frequency of afferent transmission can cause alter-ations in the descending modulatory pathways located in thebrainstem, which help to facilitate afferent stimulation. Thedeveloping cycle of positive feedback instigates the post-translational and transcriptional changes within the secondorder afferents that result in hyperalgesia and allodynia orserves to dissociate peripheral input from nociceptive trans-mission. Because the resulting pain is independent of periph-eral input, treatments aimed at the periphery are rarely suc-cessful. The absence of successful conventional therapies hasmotivated the development of novel treatment modalities.Thus, the remainder of this article will focus upon current andpotential future treatments for intractable chronic pain. Abrief introduction to endogenous analgesic mechanisms willprovide a basis for description of the classes, rationale for,and limitations of current pharmacologic treatments.

PHARMALOGICAL ANALGESIA4,38,41

Endogenous Opioid ActionsFour classes of endogenous opiates have been demon-

strated, including enkephalins, dynorphins, endorphins, andendomorphins. The enkephalins are derived from the pre-proenkephalin (PENK) transcript, with two primary splicevariants yielding [Leu5]- and [Met5]- variants. Multipledynorphin variants are produced from the prodynorphin(PDYN) transcript (e.g. A & B dynorphin, �- & �- neodynor-phin). Analgesic endorphins include �- & �-endorphin, pro-duced as splice variants of the proopiomelanocortin (POMC)transcript. Two tetrapeptide endomorphins are known toexist, endomorphin 1 & 2. Three overarching classes ofopioid receptors, at which these agents act, have been cloned:the �, �, and K receptors. A complex receptor subclassifica-tion system has been devised to explain the varying pharma-cologic effects of the opioid analgesics; however, this systemhas yet to be substantiated by cloning of the receptors. Basedon studies of binding affinity, it is thought that the endom-orphins are the endogenous ligands for the � receptor, thatenkephalins serve as endogenous ligands for the � receptor,and that dynorphins act as endogenous ligands for the K

receptor. The �-endorphin molecule has approximatelyequivalent affinities for the � and � receptor types. Eachreceptor produces a primary, acute effect through interactionswith G proteins that ultimately acts to stabilize membraneexcitability. This provides a mechanism for preventing noci-ceptive transmission.

Briefly, distribution of the endogenous opioids andopioid receptors is not equal throughout the neuraxis. Pro-duction of POMC-derivatives occurs primarily within spe-cific hypothalamic nuclei with projections to disparate sec-tions of the brain and spine. PDYN, dynorphin, neurons arepresent within the limbic system, hypothalamus, and spinalinterneurons. Endomorphins, specifically endomorphin-2, arelargely localized to the presynaptic terminal of primary af-ferents within the spinal cord and within areas of the thala-mus. Opioid receptors have been located within the periph-eral nervous system, brainstem sites of nociceptiveregulation, the limbic system, and the spinal cord.

Upregulation of several of the endogenous opioids andassociated receptors occurs at the spinal level, in models ofperipheral inflammation. PDYN, and thus dynorphin, areparticularly upregulated. This opiate is also increased inmodels of neuropathic pain. In fact, effects of dynorphin onnon-opioid receptors may affect the sensitization process andthus the presence of hyperalgesia and allodynia. Amongnon-opioid receptors, dynorphin affects NMDAR activation,potentially countering the process of sensitization describedpreviously.



Exogenous TreatmentsClinical approaches to pain treatment vary depending

on whether the underlying etiology is acute or chronic. Acutepain is commonly treated through the widespread use ofNSAIDS, opiates, or a combination of the two. NSAIDS arelargely presumed to act in the periphery by reducing thecontribution of prostaglandins to the process of peripheralsensitization. The different enantiomers of NSAIDS, R & S,may have a different effect within the spinal cord, witheffects on both SP and NMDAR. In contrast, opiates arethought to have a predominantly central effect, at the presyn-aptic terminal of first order neurons. As mentioned previ-ously, opioid receptors are upregulated in response to tissuedamage, in this case at the dorsal root ganglion, with transportto the presynaptic terminal within the dorsal horn.

Opiates are used as a first line treatment for chronicnociceptive pain, but are relatively ineffective at the controlof neuropathic pain. Tolerance and the potential for depen-dence limit application to chronic pain. To address this, andissues of CNS depression, associated with chronic use of highlevels of opiates, intrathecal approaches have been used tobathe the spinal cord in analgesic medication. This allowsanalgesic effects to be achieved, at lowered dosage and witha reduced complement of off-target effects. Nevertheless,escalating tolerance, dependence, inconvenience, and incom-plete effectiveness remain issues of importance and should beweighed carefully in considering treatment options for indi-vidual patients. The off-target effects of opiate analgesia onconsciousness, together with the poor efficacy of opiatetherapy for neuropathic pain, provide motivation for devel-opment of more anatomically and pharmacologically specifictherapies.

In light of the limited efficacy of opiates on neuropathicpain, a variety of pharmacological treatments are used. All ofthese drugs were initially developed for application to otherpathologies. Antidepressants, specifically tricyclic antide-pressants, are thought to be useful because of their preventionof norepinephrine uptake in addition to their role in theprevention of serotonin uptake. They have proven to be moreeffective in treating neuropathic pain than SSRIs, whichsolely modulate serotonin uptake. Additionally, the effects ofthe tricyclic antidepressants are almost immediate, as com-pared with their role in treating depression, which can takeweeks. Their mechanism of action may function throughstimulation of descending tracts from the Locus Ceruleus andRaphe Magnus, increasing their effects on descending mod-ulation. Evidence also exists to support an NMDAR antago-nist mechanism for tricyclics.

Anticonvulsants such as gabapentin, carbamazepine,and clonazepam are also widely used for the treatment ofneuropathic pain. Although their mechanisms are incom-pletely understood, they are known to act as membrane

stabilizers. In particular, carbamazepine is known to blockpre-synaptic Na�. Muscle relaxants, such as baclofen anddiazepam, are used in the treatment of neuropathic pain. It isspeculated that decreased skeletal muscle tone can modify thepresence of painful muscle spasms, allowing for better con-trol over pain. At a molecular level, baclofen and diazepamact as GABA agonists, likely serving as muscle relaxantsthrough an effect on spinal inhibitory interneurons. Localanesthetics have been considered due to their ability to blockindividual ion channels, thus preventing afferent transmis-sion. Agents that mimic the effect of lidocaine such ascarbamazepine, mexiletine, and tocainide may reduce chronicpain through specific inhibition of the NMDAR. No antago-nists exist that are entirely specific for the NDMAR. How-ever, potential imperfect analogues, such as TCA, ketamine,dextromethorphan, Mg2�, and phencyclidine (PCP), haveanalgesic properties. Nonetheless, the potential for severeoff-target effects exist with NMDA antagonism, as illustratedby PCP.

Although there are currently a number of pharmacolog-ical options for the treatment of chronic nociceptive pain, aseries of limitations exists for each of the options. Prolongeduse of high-dose NSAIDS, especially those not specific forCOX-2 can have deleterious effects on homeostatic tissuefunctions. Furthermore, although some evidence exists forcentral action, a lack of evidence exists to indicate effective-ness for neuropathic pain. The use of opiates, althougheffective for acute scenarios, is fraught with drawbacks forthe treatment of chronic pain. Issues of tolerance, addiction,physical dependence, depressed quality of life, and a potentialneed for slow titration to avoid side effects need to be takeninto account uniquely for each different drug. The otherclasses mentioned are primarily indicated for other conditions(e.g. antidepressants, anticonvulsants, muscle relaxants).Thus, off-target effects must be taken into close considerationfor individual patients. Local anesthetic analogues can resultin CNS depression through their lack of specificity to noci-ceptive pathways. Considerable effort is being made to pur-sue an NMDAR specific antagonist to attempt to preventoff-target effects that plague the non-specific interactions ofthe current NMDAR antagonists. Nonetheless, it is possiblethat NMDA blockade will have to occur specifically withinthe nociceptive relay system to avoid the cognitive sideeffects seen with agents like ketamine and PCP.

Cell- and Gene-Based Approaches: Expandingthe Armamentarium12

The underlying rationales for considering either cell- orgene-based therapy are similar in that both techniques pro-vide a method to simultaneously achieve anatomic and phar-macological specificity, hence minimizing deleterious off-target effects. Anatomic specificity is achieved throughstereotactic implantation. Pharmacological specificity is



achieved by the use of an appropriate cell line or transgeneconstruct. The concept of anatomic and pharmacologicalspecificity is more fully outlined in Figure 10.6. Cell linescan be selected or engineered to secrete specific agents, hencecreating microscopic biological pumps. Gene transfer can beused to induce local secretion of these agents or to alter theintracellular signaling necessary for nociception.

Cellular “minipumps” provide several possible ad-vantages. Within the context of treating neuropathic pain,cell-based approaches are ideally suited to large scaleproduction and secretion of antinociceptive or trophicpeptides. Local concentrations are raised enough toachieve a desired effect, while minimizing the potential for

off-target effects. These cell grafts can produce labilepeptide products which, by nature, have short half lives invivo. Release of such labile secretory products, in closeapposition to the desired target receptors, increases thelikelihood for achieving a therapeutic effect. Gene basedtherapies can contribute to this process of cell basedtreatment by programming an implanted cell line to secretea desired product in a process known as ex vivo genetransfer. The same end may be achieved through a purelygene-based therapy by incorporation of transgenes intoneural tissue through the process of in vivo gene transfer.As mentioned, in addition to provoking the release ofsecreted analgesic molecules, in vivo gene transfer can

FIGURE 10.6 Specificity (anatomic versus pharmacological). A, different tissue types (large ovals) with unique cell types (circles)express a complement of receptors. Although different cell types are present in different tissues, some overlap between receptorsexists. B, ablation of a tissue locally destroys cells within the range of the instrument, regardless of cell type. This approach isanatomically, but not pharmacologically, specific. C, pharmacological therapies may be specific for a given receptor type orsubtype. However, this receptor (or subtype) may be present in different tissues, leading to off-target effects. Thus, drug therapieslack anatomic specificity. D, local administration of an agent represents an approach to achieve high concentration of an agentin a local area and to prevent off-target effects that result from the activation of receptors outside of the desired field of action.Local pumps created by cellular and gene-based approaches can achieve this balance of anatomical and pharmacologicalspecificity.



also be used to neuromodulate the intracellular signaltransduction cascades that provide the architecture forafferent signal transmission.

The individual molecules that underlie nociception,neuropathic pain, central sensitization, and the developmentof chronic pain provide a host of potential targets for atailored molecular approach. Most attempts at reversing theneuropathic pain state focus on modulation of transmission atthe level of damage within the spinal cord. We will providea brief background for cell- and gene-based approaches andthe current progress reported in the context of spinal, noci-ceptive neuromodulation.

OVERVIEW OF CELL-BASEDTHERAPY8–10,12,14,18,23,51

Cell-based therapy for neuropathic pain was initiallyborne of the recognition that adrenal chromaffin cells are capa-ble of secreting a variety of neurotransmitters and anti-nocicep-tive peptides similar to those released in both descending mod-ulation and by inhibitory interneurons. In particular, chromaffincells implanted into the spinal subarachnoid space have beendemonstrated to raise levels of met-enkephalin, catecholamines,and to reduce morphine cross-tolerance when used in conjunc-tion with morphine. Preclinical testing has shown that molecularalterations within the dorsal horn accompany the changes in CSFcomposition. Markers associated with central sensitization andneuropathic pain are depressed, including NADP-diaphorase,cGMP, c-fos, and NMDAR-hypersensitivity. Furthermore, thesepromising molecular alterations within the dorsal horn are ac-companied by behavioral alterations, indicating an analgesiceffect in a variety of animal models. These include the formalinresponse, chronic inflammation, central windup, central sensiti-zation, and neuropathic pain. Administration of catecholamineand opioid antagonists during testing has helped to ensure thatthe behavioral modification occurs, at least partially, as a resultof elevated opioid and catecholamine concentration in the CSF.In contrast, recent data demonstrates that simultaneous treatmentwith NMDAR antagonists augments the antinociceptive effectof the graft. After proof of concept was established in animalmodels, initial clinical trials were implemented to test the safetyand secondarily the efficacy of this treatment modality in pa-tients with intractable, terminal cancer pain.

These Phase I and II trials highlight several criticalpractical issues in the development of this therapeutic ap-proach. Adrenal chromaffin cells harvested from cadavericdonors were used for these trials as opposed to xenogeneicalternatives in part because use of allogeneic cells mitigatespotential for graft rejection. As a primary donor source,however, cadavers are not consistently available and do notprovide a homogenous source of graft tissue.

Ex vivo gene transfer to immortalized cell lines hasbeen used to address this issue. In addition to deliveringgenes for secreted analgesics, ex vivo gene transfer can be

used to deliver genes that prevent replication. This strategyhas been used to regulate graft proliferation. Controlledproliferation provides large quantities of homogenous graft-able cells. Unlike cells harvested from a donor, homogeneousgrafts have a more standardized and predictable impact. Celllines are inherently immortalized, which allows long-termmaintenance under in vitro conditions. This characteristicprovides the flexibility of prolonged ex vivo storage withlittle concern for viability. This technology has also allowedincorporation of transgenes into cell lines of interest, provid-ing a mechanism to specifically control peptide production ina desired cell type. Finally, techniques for semipermeablegraft encapsulation have been researched to isolate xenogeniccell sources from the immune system and prevent uncon-trolled graft migration.

Ex Vivo Gene Transfer: Control of CellProliferation

A variety of immortalized cell lines have been used aspotential analgesic grafts. To establish proof of principle,immortal cell lines with known secretion of antinociceptivetransmitters have been used in preclinical animal models. Forexample, the PC12 cell line, which is an adrenal medullarytumor line, secretes adrenergic neurotransmitters. Non-adre-nal lines have also been used including; B16, NB69, AtT-20,Neuro2A, AtT20/hENK. These have been experimentallyused for introduction of hardy, immortal cells in animalmodel transplant. These immortal cell lines often secretepeptides with potentially analgesic properties.

The ability to passage these cells provides a readysource of homogeneous therapeutic cells. However, immor-talized cell lines carry a high risk of tumorigenicity. Byintroducing a regulated oncogenic element into a cell line,cells will undergo substantial ex vivo proliferation, which canbe prevented after transplantation. One such method, condi-tional immortality, uses cells transfected with a temperaturesensitive version of the Tag oncogene. This oncogene isattained from Simian Virus 40 (SV40). When grown attemperatures below approximately 39oC, the cells continue toproliferate in an undifferentiated state. When cultured orimplanted in tissue that remains at or above this temperature,differentiation occurs with a resultant increase in peptideoutput. The still present, though repressed, oncogene doesleave a potential for in vivo proliferation. Furthermore, thisresidual oncogene expression suppresses the secretion ofanalgesic peptides. Reversible immortalization has been de-veloped to address these shortfalls by entirely removing theoncogenic sequence from the transfected cell once differen-tiation is desired. In this way, considerable ex vivo passagingcan be accomplished while still providing a mechanism tocontrol the potential for graft tissue proliferation. The Cre/loxsystem provides one method to achieve reversible immortal-ization. Cre recombinase recognizes the loxP sequence, cut-



ting DNA at this point, recombining it with another loxP site.By flanking the oncogene with loxP sites, Cre can be used toclip out the oncogene. The Cre protein can itself be deliveredas a gene under the control of an inducible promoter. There-fore, by inducing Cre expression in the cells containing theforeign genes, the oncogene is removed.

Ex Vivo Gene Transfer: Transgenic PeptidesMany of the cell lines used for grafting do not naturally

secrete analgesic peptides, but are chosen for other advanta-geous qualities. Under these circumstances, the ability toproduce neuromodulatory peptides may be conferred throughthe processes of gene transfer. It is advantageous in thesecases to use cell lines with a secretory or neuroendocrineorigin to ensure the presence of an appropriate intracellularsecretory apparatus. Gene transfer can also be used to aug-ment existing antinociceptive peptide production. For exam-ple, �-endorphin is produced by the immortal cell line,AtT20. A variant, AtT20/hENK, has been transfected withthe gene for preproenkephalin (PPENK). In this cell line, bothenkephalin and �-Endorphin, the natural secretory productsof AtT20 cells are produced. Additionally, when neuron-committed progenitor cells of the NT2 cell line were engi-neered to express PPENK and intrathecally implanted, anti-nociceptive effects comparable to those attained withchromaffin cell implant were obtained. Secretory transgenesintroduced for the treatment of neuropathic pain are notrestricted to the classic antinociceptive peptides. A rat neu-ronal cell line RN33B, was both conditionally immortalizedand transfected ex vivo with the gene for brain derivedneurotrophic factor (BDNF). In a chronic constriction injury(CCI) model of chronic pain, both immunohistological andbehavioral measures indicated that the BDNF served ananalgesic role throughout the follow-up period. Recently, ithas been demonstrated that both reversible immortalizationand the presence of a secretory transgene could be simulta-neously incorporated into a transplanted cell line. Vectors foreither met-enkephalin or proenkephalin expression caused areduction in dorsal horn c-fos. These experiments point to thefeasibility of creating a homogenous cell line, with controlledreplication, capable of secreting desired peptide products.Examples of other transgenes that have been tested in variousmodels for the treatment of neuropathic pain include: galanin,GABA, and POMC.

Methods of Immune Surveillance ProtectionGrafts not derived from the host have the potential to

provoke an immune response resulting in graft destructionand secondary injury to neural structures. Transplants havelargely targeted the subarachnoid space or the spinal paren-chyma. In these models, cells are placed into an immuno-privileged site. The subarachnoid space and CNS permitaccess to fewer circulating immune cells in comparison with

other targets for cell or organ transplant. Although immuno-privileged, the potential for rejection remains, prompting theuse of immunosuppressants in some experiments. Many ofthe cell lines studied are xenogeneic, further increasing thelikelihood of recognition by the immune system.

Immunoisolation of the transplanted cells is an alterna-tive method to address this issue. Before transplant, grafts areencapsulated in a semi-permeable, polymeric membrane. Theporosity of the polymeric membrane is designed such thatwaste products and the secreted neuromodulatory productmay diffuse away from the encapsulated membrane whilenutrients can diffuse towards the encapsulated cells. At thesame time, this system prevents recognition by antigen pre-senting cells. Multiple encapsulation constructs are currentlyused, with different methods of production. Both sphericaland cylindrical shapes have been produced. Preclinical test-ing has provided evidence for prolonged in vivo cell viabilityand capacity for secretion, extending out beyond 500 days.Furthermore, continued catecholamine and opioid secretionhas been demonstrated at pre-implant levels up to 3 monthsfollowing transplant. Recent clinical trial evidence indicatesthat immunoisolation of implanted cells will prevent theconcomitant need for immunosuppressants and reduce thelikelihood of implant rejection.

Immunoisolation can also be achieved by minimizingthe antigenicity of implanted graft cells. In the use of primarycell graft implants, this can be partially achieved by ensuringa purified implant. In use of adrenal chromaffin cells, inad-vertent incorporation of unnecessary tissue parenchyma willsignificantly increase the antigenic load of the implantedgraft. Another method that has been used to circumventimmune detection is the use of antigenically immature cellsas a means to prevent substrate detection. This can be ac-complished by using stem cells, progenitors, or fetally-de-rived cells for transplant.

Neural Stem Cell and Progenitor CellContributions

Implantation of neural stem cells represents one newlyresearched avenue of cell-based therapy. Implantation ofneural stem cells into the spinal cord has been used inpreclinical experimentation for treatment of spinal cord inju-ries (SCIs). This approach has been shown to contribute tore-establishing functional connectivity and myelination ofdamaged fiber tracts. Establishing reconnection of severedfiber tracts is accomplished either directly through differen-tiation into nerve cells or indirectly through differentiationinto supporting, trophic cells (oligodendrocytes) that help toestablish an appropriate niche for regeneration. Remyelina-tion is aided by differentiation into oligodendrocytes, allow-ing direct fiber protection and preservation of signal conduc-tion. The predominant pattern of differentiation is thought todepend upon the trophic milieu surrounding implanted stem



cells. Evidence now indicates that this research may haveramifications for the treatment of neuropathic pain.

A recent study performed by Hoffstetter et al. comparedthe effects of neural stem cells (NSCs), NSCs pretreated with atranscription factor (Ngn-2), and a control group, to determinewhich preparation yielded the most functional restoration in a ratmodel of SCI. The transcription factor, Ngn-2, helps to direct thelineage differentiation of the NSC population. Whereas theuntreated NSC population aided in the re-establishment of fibertract connectivity and in remyelination, a significant degree ofallodynia also developed that was not present in either thecontrol group or in the NSC�Ngn-2 group. Additionally, bothre-connectivity and sensation was improved to a greater extentin the NSC�Ngn-2 group without evidence to support concom-itant development of allodynia. Additional fiber sprouting oc-curred in the untreated NSC group that was not present in theNgn-2 � NSC group. This was attributed to greater astrocytedifferentiation in the untreated NSC group and mentioned as thelikely cause of allodynia development in the NSC-only group.These results have multiple implications for the treatment ofneuropathic pain.

Current approaches to treatment of neuropathic painfocus on reversing an already present condition. In contrast,these results provide initial data to support the concept thatinjuries or interventional procedures associated with devel-opment of neuropathic pain could be treated prophylacticallywith stem cell-based therapies that are aimed at preventingneuroma formation. Appropriate implementation of stemcell-based therapies, after injury, but before development ofneuropathic pain, could help to re-establish or improve func-tional connectivity while minimizing the potential for aber-rant axonal sprouting. By helping to prevent a repair responsegone awry, and a subsequent over-excitatory input to thedorsal horn, central sensitization could be prevented fromoccurring. This represents a potential means to treat neuro-pathic pain of a defined, injury-oriented etiology. Further-more, to achieve a synergistic effect, stem cell-based thera-pies could be used in concert with other cell-based, genetransfer-oriented approaches. This could be particularly use-ful in terms of providing the appropriate chemical, trophicmilieu necessary to ensure long-term graft viability withneural stem cells and progenitors. In the present, however, theallodynia associated with naı̈ve NSC implantation suggests aneed for greater understanding of the differentiation potentialof an implanted cell type so as to prevent exacerbation ofneuropathic pain. Furthermore, it indicates a need to morefully understand the factors that drive differentiation towardsparticular lineages.

CELL-BASED CLINICAL TRIALS8,12,24,44,51

As mentioned above, adrenal chromaffin cells were thefirst and remain the most studied cell type for the treatment ofneuropathic pain. To date, chromaffin cell implantation rep-

resents the only cell-based therapy to have undergone clinicaltrial testing. These trials were based on preclinical testing,which largely used grafts in animal models to measureantinociceptive-related effects relating to both biochemicaland behavioral markers. Initial clinical trials designed toexamine the safety and efficacy of both unencapsulated andencapsulated adrenal chromaffin cells have been completed.

Unencapsulated Chromaffin Cell TrialsThe initial trial of cell-based therapy for neuropathic

pain consisted of two patients. An analgesic effect was notappreciated. However, in this trial, graft material for individ-ual patients was heterogenous, having been attained frommultiple donors for individual grafts. Furthermore, implantwas performed on a same-day basis following tissue harvest,with no attempts to ascertain graft tissue viability.

A larger unblinded, uncontrolled trial (n�5) was sub-sequently conducted by Winne and Sagen between 1991 and1993. Terminal cancer patients with intractable pain werechosen for this trial. Patients included in this trial showedsystemic side effects to opioid treatment, but had not yet beentreated by alternative approaches, including intrathecal mor-phine. Adrenal chromaffin cells were obtained from cadav-eric donors. Explants from the adrenal medulla were culturedin vitro for approximately 1 week to ensure explant viability.Patients were given immunosuppressant treatment (cyclo-sporine) before implantation and for 2 weeks after implant toprevent graft rejection. Within 1 to 1.5 months, four out of thefive patients experienced an improvement in pain, as indi-cated by visual analog pain scores. Additionally, three ofthese five patients indicated significant reductions in: painscore, analgesic consumption, and increases in activity. In-creases in CSF opioid and catecholamine levels over pre-implant levels were noted. However, in all cases, largevariability was present, preventing the ability to ascertainmore than a trend.

A Phase II trial conducted by Lazorthes et al. enrolled15 individuals, also with terminal, intractable cancer pain. Incontrast to the previous trial, inclusion criteria required thatpatients be refractory to systemic opioid treatment owing toan accumulation of dose-related side effects. In all patients,the pain condition had progressed such that intrathecal opioidadministration had been attempted before graft implant in allparticipants. Of the patients in the trial, five no longerrequired intrathecal morphine injections, two required a less-ened dose, and five had stable morphine requirements. Ben-efit was not demonstrated for the remaining three patients.The lack of effect was hypothesized to be due to poorparticipant selection, insufficient quality and quantity of im-planted graft tissue, impaired allograft function, immunolog-ical graft rejection, and distance between implant engraftmentand affected area leading to insufficient local titers of antino-ciceptive peptides. Autopsies performed on two patients that



survived over a period of 1 year indicated prolonged graftsurvival. Using the rationale that the CSF is immunoprivi-leged, grafts were neither encapsulated nor HLA typed to therecipient. Lymphocytosis was noted in 75% of the trans-planted allografts. However, no effect was noticed on CSFmet-enkephalin levels, which the authors interpreted as pro-viding indication that the grafts were tolerated, at least for theduration of follow-up period for this trial (approximately 4.5mo). The only reported adverse events were associated withimmunosuppressive treatment and intrathecal morphine ad-ministration, when required. Ultimately, tolerance of the graftand the lack of observed side effects helped to establish safetyand feasibility of this treatment modality. Additionally, theoverall reduction in concomitant opioid therapy providedimpetus to further explore this and related cell therapies inlarger, placebo-controlled trials.

Encapsulated Chromaffin Cell TrialsAs mentioned previously, the use of encapsulation

resulted from the recognition that primary adrenal cell graftswould require containment and immunoisolation. To date,two trials have been attempted using encapsulation of xeno-geneic adrenal medullary chromaffin cells. Concomitant im-munosuppressant therapy was not used in either trial.

The initial Phase I trial was designed to test safety ofthe encapsulated cell transplant intervention. To this end, allsymptoms associated with the procedure were minor, self-limited, and were related to the implantation. Further, a needfor depressed analgesic usage was noted, providing impetusfor a larger, controlled trial. Immunosuppressants were notused in this trial. The second trial was prematurely haltedowing to an inability to establish efficacy of the proposedtreatment.

CONFLICTING RESULTS: CAUTION FOR ATEMPERED APPROACH16,28–30

It should be mentioned that this negative result is notisolated. Several preclinical studies have failed to reproduceantinociceptive results with bovine chromaffin implantation.It has been postulated that the lack of efficacy in the trial andthe similar preclinical results may be due to the use of aninsufficient number of cells. Thus, recent preclinical studieshave examined the use of chromaffin implants when com-bined with intrathecal administration of an NMDAR antago-nist. Results indicate that the analgesic effects of the twomodalities in tandem could provide synergistic effects. Aug-mentation of the analgesic response, using this methodology,could help to produce a clinical effect, even if a sub-thera-peutic cell number is implanted.

OVERVIEW OF GENE-BASED THERAPY14,21,31,39

As previously described, overlap exists between thetherapeutic indications for cell- and gene-based therapies.

Furthermore, ex vivo gene transfer represents a hybrid of thetwo techniques. However, gene transfer provides a widerplatform from which to design interventions for the treatmentof neuropathic pain. In vivo gene transfer techniques can befurther stratified based on the vectors used for gene delivery.In in vivo gene transfer, genes are inserted directly into thecells of parenchymal tissue. As such, different routes havebeen devised by which to reach these tissues while simulta-neously minimizing the invasive nature of the therapy. Vec-tors may be injected intrathecally, from which point thegenetic material of the vectors are incorporated into adjacentparenchyma. Intraparenchymal vector delivery refers to di-rect injection of vector material into an anatomically specificportion of parenchyma. Finally, remote delivery refers to aspecialized form of delivery in which neurotropic viruses areutilized to deliver genetic material to a precise anatomiclocalization via retrograde axonal transport.

To achieve expression, transgenes must be inserted intoan expression cassette. The expression cassette includes apromoter, which drives expression. Expression of the genealso requires a polyadenylation sequence (pA) placed at the3� end of the gene. A variety of promoters exist that can driveexpression constantly or in a regulated fashion. Some pro-moters are specific for individual cell types and even neuronalsubpopulations, restricting the expression to specific targetcells.

Current experimental evidence for the treatment ofdifferent facets of neuropathic pain will be presented in termsof the mechanism of vector delivery following a brief intro-duction to the vector types utilized to mediate in vivo genetransfer, which can be broadly classified as viral or non-viral.

Vector System: Non-viralIn a comparison of vector types, use of nonviral vectors

provides the distinct advantage of simplicity, exemplified bya lack of potential for pathogenicity and minimized immu-nogenicity. The simplest approach is to use naked plasmidDNA, attaining electro-permeabilization with specifically de-signed intrathecal probes. A primary limitation of this meth-odology is consistently short expression of the engineeredtransgene.

In attempts to increase transgene uptake and expres-sion, various attempts at coating the genetic material havebeen attempted. Both liposome-mediated (lipoplex) transfer,using cationic lipids, and polymer-mediated (polyplex) trans-fer, using hydrophilic polymers, have been attempted. Whenused in conjunction, the vector is termed a lipopolyplex.These coatings condense the genetic material partially byisolating the anionic charges present on the structural phos-phate moieties. Peptide or protein conjugates with DNArefers to a molecular conjugate. When designing a non-viralvector, the particular coating material and proteins whichmay be conjugated to this material are carefully chosen to



maximize the processes of vector binding, endosomolysis,cytosolic trafficking, and nuclear entry.

Vector binding can either be receptor mediated ornon-receptor mediated. In efforts to achieve receptor medi-ated binding, multiple ligand types have been coupled togiven coating materials. Polyplexes using poly-L-lysine(PLS) have been used to couple a variety of ligands. Addi-tionally, molecular conjugates can and have been used forcoupling antibodies designed to recognize specific receptorswith vectors. Non-receptor binding is facilitated in bothpolyplexes and lipoplexes as a result of their positive charges.Evidence is present to suggest that this binding occurs, atleast partially, to negatively charged surface proteoglycans.Molecular conjugates have also been shown to use proteo-glycans for either primary binding or as co-receptors.

The mechanism by which a particular nonviral vectorbinds to the cell surface determines whether or not it will beincorporated into a lysosomal or endosomal degradation path-way. Large vectors (approximately 500 nm) seem to bemaintained within uncoated vesicles and are ultimately notdegraded through endosome fusion with lysosomes whereascoated endosomal vesicles (approximately 80–100 nm) aredegraded through this fusion process. Regardless of whichcategory a vector falls under, endosomolytic mechanismsmust be present to assure acceptable gene expression. Mul-tiple mechanisms exist whereby nonviral vectors can releaseDNA; pore formation, membrane flip-flop, or proton spongemediated osmolysis. Other putative mechanisms take advan-tage of a combination of these processes. Additionally, genetransfer efficiency can be modulated by the use of agentswhich alter the stability of cytoskeletal microtubules.

The presence of a nuclear localization sequence (NLS)helps to ensure that the transfected genetic material willactually reach the nucleus. At the nuclear membrane, itpromotes aggregation of a pore complex. Several proteinsand peptide sequences have been used as putative NLS. Inlipoplexes and polyplexes that have not undergone molecularconjugation, cell division seems to significantly increasetransgene uptake and expression in at least some cell types.However, dependence on cell division inherently targets genedelivery to non-neuronal populations.

Nonviral vectors, engineered to tightly hold geneticmaterial, may ultimately inhibit expression as noted by anexperiment in which both naked plasmid DNA and lipoplexbound DNA were both injected into nuclei. In contrast,however, early exposure of the transfected genetic materialwithin the cytosol can also result in poor transgene expres-sion, due to nuclease dependent degradation.

Vector System: ViralViral vectors provide the advantage of increased trans-

duction efficiency. Additionally, some strains have inherentneural tropism. Recognizing a need to minimize the possibil-

ity for in vivo proliferation and to minimize the potential forimmune recognition, vectors have been attenuated through aprocess of removing critical replication-specific genes. Inaddition, inessential accessory genes have been removed insome strains to minimize the potential for immune recogni-tion and to maximize free space for transgene incorporation.Gutting viral vectors of non-essential genes prevents expres-sion of non-self peptides, but requires the presence of helperviruses during incubation to provide the genomic contentnecessary for replication. In this manner, potential for cellsurface expression of foreign peptides via the presence ofMHC Class I peptides is minimized.

The general anatomy of the vectors to be considered aresimilar. An outer lipid bilayer envelope may or may not bepresent. When present, it is studded with proteins that aid intarget recognition and uptake. If the bilayer exists and ifproteins necessary for neural tropism are not naturallypresent, pseudotyping techniques may be used to add theseproteins. In the vectors with an outer bilayer, a tegumentconsisting of necessary enzymes and proteins surrounds anucleocapsid which may contain nuclear localization signalsand contains the genetic material. At minimum, all viralvectors contain a nucleocapsid. If they only possess a nucleo-capsid, then receptor binding proteins will be present whichcan also be pseudotyped to target gene delivery. Viral vectorsmay be categorized as being either neurotropic or non-neurotropic, a classification which determines suitability forremote viral delivery. Such tropism reflects the presence ofviral surface proteins that facilitate cell type specific uptakeand retrograde axonal transport.

Herpes Simplex Virus (HSV) and Rabies Virus (RV)are neurotropic viruses. HSV has been extensively engi-neered for use as a vector. This tropism is, at least in part,conferred by the presence of glycoprotein-mediated bindingto neural cell surface proteoglycans. Both are capable ofanterograde and retrograde axonal transport, in a strain de-pendent manner. This process allows subcutaneous adminis-tration to result in dermatome-specific DRG expression. Ad-ditionally, they are capable of creating latent infections inpost-mitotic cells. The HSV-1 genome is 152kb allowing forease of manipulation in transgene incorporation. Herpes vi-ruses attenuated by ‘gutting’ are known as amplicons. Byvirtue of the fact that fewer viral proteins are produced, andthus displayed, on MHC I surface proteins, this processreduces the potential for immune recognition and uncon-trolled proliferation, but complicates vector construction.Deletion mutants have been optimized to prevent reversion towildtype while preserving portions of the original genome.

Lentiviruses (LV) represent a unique subcategory ofretroviruses (RV). Like HSV, they are capable of infectingnon-dividing cells because they contain enzymes that providefor nuclear docking and energy-dependent transport acrossthe nuclear membrane. HIV is a well known member of this



family, and most lentivectors are derivatives of HIV-1. Theextensive understanding of HIV virology motivated the earlydevelopment of this vector system. Lentiviral particles havebeen both attenuated and modified to increase the range oftissues susceptible to infection through pseudotyping. Lenti-viruses have been modified over successive viral generationsto enhance attenuation and safety. The most recent iterationscontain only three original genes, while retaining transduc-tion potential for neurons in vivo. Persisting concerns for invivo reversion to wildtype have been addressed by use ofnon-human lentiviruses (e.g., EIAV and FIV) and through theuse of self-inactivating lentiviruses, attained through deletionof viral genome LTRs.

Adenovirus (AV) is a non-enveloped 36-kb dsDNAvirus that is useful as a delivery vector, in part, because of itshigh transduction efficiency in multiple tissue types, irrespec-tive of cell division potential. An additional positive attributeis a lack of association with any human neoplastic patholo-gies. Viral uptake is partially mediated through binding to theMHC Class I receptor, followed by subsequent endocytosis.Upon internalization, the genetic material, still partiallycoated, is localized to the nucleus. However, viral geneticmaterial does not integrate into the cellular nuclear material.A mounted immune response and transgenes driven by pre-dominately short-acting viral promoters have traditionallylimited the length of experimental effects to the order of a fewweeks. A newer generation of “gutless” AV vectors lack allnatural viral sequences except those necessary for DNApackaging. As with other gutless vectors, the likelihood thatan immune response will be mounted is significantly reduced,as nearly all potential viral antigens are removed. Further-more, by gutting the AV vector it gains the ability to packagelarge therapeutic transgenes and complex expression systems.

The adeno-associated virus (AAV) is a non-enveloped4.7kb ssDNA virus that is not associated with any humanpathologies. AAV strains are not naturally pathogenic inhumans and are naturally attenuated, requiring the presenceof a helper virus to provide the complementary genomicmaterial necessary for replication. In vivo, it incorporatesdirectly into the host genome. This feature may be responsi-ble for the long transgene expression that has been noted tolast indefinitely in some cases. In wildtype form, the maxi-mum transgene size that can be incorporated is relativelysmall. Recent efforts, focusing on heterodimerization of viralparticles, have showed promise for potentially doubling thesize of transgene incorporation. Like the AV vector, AAV hasthe capability to infect a wide range of dividing and non-dividing cell types. Pseudotyping has resulted in the creationof multiple serotypes of AAV in an effort to optimize theviruses for different experimental approaches.

In summary, gene transfer can be achieved with viral ornonviral systems. Whereas the nonviral systems have lesspotential inflammatory side effects, they trigger very transient

and limited gene expression. Viral vectors are more efficientbut also more complex. First generation vectors, includingHSV and AV, have a limited duration of gene expression,whereas LV and AAV vectors induce long lasting expressionwith minimal inflammatory consequences. Although HSVhas inherent tropism to primary sensory cells, pseudotypingpromises to deliver enhanced neurotropism to lenti and AAVvectors.

EXPERIMENTAL EVIDENCE FOR IN VIVO GENETRANSFER (6,11,15,17,20,25–27,33,34,39,45,46,50,52–55)

A variety of approaches have been used to apply genetransfer to models of pain. Approaches differ with respect tothe transgene selected, mode of delivery, vector type, andmodel of pain. Specific therapeutic transgenes have beenexplored for inflammatory, nociceptive, and neuropathicpain. Much of the work cited in this section is new data andso the results are currently published as abstracts.

Intrathecal DeliveryIntrathecal injection of both non-viral and viral vectors

have been used. Lin et al. have previously shown that elec-troporation following intrathecal incorporation of naked plas-mid DNA encoding POMC induced pial transduction in a ratmodel. Opiate precursor peptides have proven particularlyeffective in models of nociceptive pain. In this model, theywere able to achieve a transient anti-nociceptive effect. Morerecently, this group has reported success in achieving controlof this anti-nociceptive response using a similar POMCplasmid construct modified to incorporate a transrepressorsystem that is capable of depressing POMC expression in thepresence of doxycycline. After characterizing initial �-endor-phin expression and markers of antinociception, a reductionin both antinociception and �-endorphin levels was demon-strated to vary in a doxycycline dose-dependent manner.

An early attempt to develop an analgesic viral vectoruse first generation AV vectors, encoding �-endorphin, in-jected into the intrathecal space of rats. Following the pre-dominate infection of adjacent pial cells, a temporary reduc-tion in hyperalgesic markers was noted. Subsequent inductionof an inflammatory response limited the duration of transgeneexpression. Through pial delivery of genes for analgesicpeptides, these approaches bathe the intrathecal space inanti-nociceptive peptides.

Delivery of anti-inflammatory cytokines seems to ef-fectively inhibit the nociceptive response in models of in-flammatory pain. IL-2 is thought to possess both central andperipheral analgesic effects, due in part to interactions at the� receptor. Yao et al. injected intrathecal plasmid DNA withand without lipofectamine to transfect the spinal cord. mRNAand protein were detected in the pia mater, DRG, dorsal horn,and sciatic nerve. Behavioral measures of analgesia indicatedan effect lasting six days. Subsequent use of an adenoviral



vector encoding IL-2 resulted in similar anti-nociceptiveeffects lasting 3 to 4 weeks. Milligan et al. have used multipledelivery systems to achieve in vivo production of IL-10 andsubsequent release into the intrathecal space. IL-10 waschosen for its ability to downregulate the release of TNF-�,IL-1, and IL-6. Furthermore, it has the capability to directlyaugment other anti-nociceptive processes. Intrathecal injec-tion of an AAV vector caused infection of spinal meningealcells, with consequent elevation of intrathecal (IL-10). Usinga hybrid CMV enhancer/chicken � actin promoter, analgesiawas seen for a period of approximately 1 week, transientlypreventing and reversing neuropathic pain in rat models. Useof different serotypes and/or longer acting promoters wasposited as a potential mechanism for achieving a longerduration of analgesia. In other work, Sloane et al., haveexperimented with the use of non-viral vectors in an attemptto achieve longer term analgesia. Recently published resultshave indicated success in use of non-viral vectors to achievean increased duration of effect. Dual injections, when appro-priately spread (between 5 and 72 hrs), were able to maintainlong-term behavioral reversal for a period of up to 40 days.Recently, Milligan et al. have examined the use of nakedplasmid DNA, plasmid DNA coated with PEI, and plasmidsencapsulated within PLGA. Single injections were noted toinduce reversal of allodynia for 3 to 6 days while appropri-ately spaced dual injections resulted in 40 or more days ofreversed allodynia in the abovementioned vector systems.Labeled PLGA is currently being used to determine the depthof spinal vector penetration. While these results are promis-ing, they have only been published in abstract form at thistime.

The delivery of small inhibitory RNAs (siRNA) has re-cently emerged as a means to suppress expression of specificgenes. As an approach to counteracting inflammatory pain, Linet al. have recently shown that intrathecal introduction of siRNAspecific to a subtype of the PGE2 prostaglandin receptor, theEP4 prostanoid receptor, is capable of reducing inflammatorypain without evoking an immune response. Concurrently,mRNA and protein levels were demonstrated to be significantlydiminished within the DRG. As with the IL-10 data, these resultshave only recently been demonstrated and remain published inabstract form. Additionally, a sole injection of siRNA against theN2B subunit of the NMDA receptor by Tan et al has recentlybeen shown to result in a diminished pain response. The reversalwas maximal at 3 days after injection for reduction in mRNAlevels and at 7 days for maximal protein subunit reduction.

Remote DeliveryRemote delivery represents a particularly powerful

mechanism by which to achieve neuromodulation as itprovides a means to deliver genes to the DRG neurons viaa subcutaneous injection. This process is largely accom-plished by the use of viruses that are either naturally

neurotropic or which have been pseudotyped to gain neuraltropism. In the wildtype form, subcutaneous inoculation isfollowed by several rounds of amplification within theadjacent epithelium. Once sufficient titers are present, theviral particles are taken up into primary afferents. Follow-ing retrograde transport, a primary infection is established.A latent neural infection is facilitated by alteration of theviral genome, as briefly described below. Vectors derivedfrom neurotropic viruses like HSV, do not replicate, butmaintain the capacity for uptake and retrograde delivery toDRG neurons. AV and AAV are capable of remote deliv-ery to a lesser extent. Pseudotyping of lentivectors hasachieved levels of remote gene delivery similar to that seenwith HSV. Some pseudotyped vectors retain the capabilityto then undergo anterograde transport into the dorsal horn,participating in trans-synaptic spread.

Pohl et al. initially demonstrated the ability to achieveHSV mediated PPE delivery to the DRG. In this vector, theviral thymidine kinase sequence was replaced by the PPEsequence. Wilson et al. extended this work, determining thatbehavioral responses (foot withdrawal) were stunted whenrats treated with this HSV-based viral vector were exposed tonoxious chemical stimuli (capsaicin or dimethyl sulfoxide) asopposed to controls. They further reported that this reversalcould be antagonized by naloxone administration. Furtherexamination by Goss et al. has shown elevated production ofproenkephalin in the DRG with a reduction in pain behaviornoted in the formalin model. Goss et al. also noted that,although the effect had disappeared 4 weeks post-inoculation,re-inoculation at that time resulted in the return of anti-nociceptive behaviors, when tested the following week. Morerecently, Wolfe et al. have used the HSV vector as a vehicleto drive overexpression of endomorphin-2 via a similar re-mote approach. Success in achieving multiple markers ofanti-nociception, included suppressed mechanical allodynia,thermal hyperalgesia, paw edema, and differential hind pawweight bearing in a model of inflammatory pain. Reducedhind paw withdrawal was noted in both acute and long-termphases of the formalin response. Reduced mechanical allo-dynia and thermal hyperalgesia were noted in the spinalligature model of neuropathic pain.

Each of these experiments demonstrate that overex-pression of anti-nociceptive peptides modulates networksignaling to achieve analgesia. The use of anti-nociceptivepeptides provides one means to modulate network activity.Other avenues have been explored focusing at the level ofthe individual neurotransmitters. Neuropathic pain second-ary to partial nerve injury results in a loss of GABAergictransmission. To replace lost GABA transmission, Hao etal. constructed HSV vectors for the delivery of the gluta-mate decarboxylase (GAD) gene. This vector triggersproduction of GABA in the DRG. In the neuropathic painmodel of L5 spinal nerve ligation, rats exhibited signs of



mechanical allodynia (e.g., reduced latency to withdrawalfrom mechanical stimuli) and thermal hyperalgesia (e.g.,reduced withdrawal to heat stimuli). Subcutaneous inocu-lation resulted in a reduced mechanical allodynia, begin-ning 1 week after injection. A peak in mechanical thresh-old was achieved two weeks post-inoculation with anincreased threshold persisting for 5 to 6 weeks. Tolerancewas not noted as re-inoculation at 2 months resulted in asimilar allodynia recovery profile. Thermal hyperalgesiawas alleviated beginning at 1 week after inoculation,peaking between Weeks 2 and 3, and persisting for 6weeks. Improvement in behavioral responses was mirroredby a block in induction of c-fos or phosphorylation ofERK1/2 that occurred in control rats.

In addition to driving overexpression of either peptidesthat promote analgesia or that promote inhibitory transmis-sion, other methods are currently being pioneered that at-tempt to modulate the internal architecture of the network. Inone such method, Yeomans et al. attempted to knock downthe NaV1.7 Na� channel present in primary afferents. Theauthors attempted to knock down this receptor because it hasbeen noted to be upregulated in inflammatory pain states andcould thus be a contributor to the process of central sensiti-zation and the development of chronic pain. After use ofremote delivery techniques, the investigators were able todetect a decrease in the prevalence of NaV1.7 Na� channelsvia immunohistochemical techniques and a reduction in in-flammatory-related hyperalgesia, providing impetus for fur-ther research. Another technique currently under investiga-tion refers to repression of gene expression through the use ofZinc finger Protein Transcription Factors. Jouvenot et al. havereported success in the use of Zinc-Finger protein transcrip-tion factors to knockdown the TRPV1 receptor, TRK-Areceptor, and Nav1.8 channel. The TrkA receptor has beenshown to be upregulated in chronic pain states after spinalcord injury, whereas pharmacological blockade of theTRPV1 channel and reduction in the Nav1.8 channel havebeen correlated with inhibition of neuropathic pain. Theywere able to achieve gene repression at the mRNA level andof protein levels as well. More recently, Tan et al. havesuccessfully used an HSV-based vector to drive gene repres-sion of the TrkA receptor and the Nav1.8 channel.

Techniques using remote delivery as a route of admin-istration have also focused on treating the root causes of somespecialized types of pain. Fink et al. have previously shownthat neurotrophic factors can be used to exert a neuroprotec-tive effect in rodent models of streptozocin-induced diabeticneuropathy. Recently, Chattopadhyay et al. reported thatsubcutaneous inoculation of HSV vectors carrying the EPOgene can exert a neuroprotective effect. Two weeks afterdiabetes induction, the animals were inoculated. Markers ofneuropathy were tested 1 month later. At this time, the

inoculated mice retained sensory nerve amplitude, preserva-tion of thermal nociception, and autonomic function.

SUMMARY AND FUTURE DIRECTIONSNociception results from an extremely complex neural

processing system that segregates the various components ofan adaptive pain response. Roughly, this system includesmechanisms for: spatial and temporal localization, escapemotivation, future avoidance of injury through efficient mem-ory formation, and autonomic accompaniment of the escaperesponse. To function effectively as a whole, efficient sub-systems exist to regulate the incoming nociceptive signals.Within the periphery and at each synaptic relay point, thisregulatory process is subserved by a variety of specifictransmitters, ion channels, and receptors that underlie andregulate transmission.

Neuropathic pain results when this complex system ismalfunctioning due to aberrant signaling stemming fromintrinsic neural pathology. The shift to chronic neuropathicand nociceptive pain results from changes in specific molec-ular mechanisms that underlie normal nociceptive transmis-sion. The alteration in the function of these molecules, par-ticularly through peripheral and central sensitization, canuncouple pain from peripheral injury or damage. As such,chronic pain often ceases to be amenable to treatment strat-egies that focus on structural disease of the body. It is criticalfor neurosurgeons to grasp this mechanism and help theirpatients to understand it as well so as to prevent the expenseand sequelae of unnecessary operations.

Although a variety of distinct pharmacological treat-ments exist for inflammatory, neuropathic, and nociceptivepain, all are subject to off target effects that can complicatetheir use. These effects exist because the target receptors mayplay different functional roles in different anatomical loca-tions. Pain surgery can provide improved specificity by tar-geting specific anatomical targets in the pain processingsystem. However, existing approaches lack the specificity toaffect individual receptor systems. Because unique siteswithin the nervous system may have multiple functions en-coded by different receptor systems, existing neurosurgicalapproaches also lack the required specificity. In addition,while the application of implanted stimulators has obfuscatedthe need for ablation in many cases, this technology requiresprosthetics which are subject to failure, infection, and break-age.

Cellular and molecular approaches have the capacityto target specific molecules within specific anatomicallocations. Therefore, they have the potential for improvedfunctional specificity over existing approaches. Moreover,as biological interventions, they are not subject to the samemechanisms of failure as implantable prosthetics. Cellularapproaches provide a means for the creation of micro-scopic pumps of specific analgesics that can integrate into



the tissue of a given nociceptive relay. Gene transfer canbe applied to control the secretion and replication of thesemicropumps.

Direct gene transfer can alter the function of existingnociceptive neurons either by inducing the secretion of anal-gesic mediators or by affecting intracellular function. Newtechnology has made it possible to introduce new genes,increase the expression of existing genes, or silence unwantedgenes. A variety of vector types, promoters, vector coats, anddelivery strategies exist to tailor gene delivery. Finally, spe-cific therapeutic transgenes have emerged that prove effectiveat impacting the molecular processes of different pain sub-types, including inflammatory, neuropathic, and nociceptivepain.

Future development will require longer lasting vectors,homogeneous grafts, control of the immune response andgene expression. The vast number of specific moleculesimplicated in nociception and sensitization provide an en-compassing array of potential targets for the development offuture molecular therapies.

REFERENCES1. Aguggia M: Neurophysiology of pain. Neurol Sci 24 [Suppl 2]:S57-

S60, 2003.2. Almeida TF, Roizenblatt S, Tufik S: Afferent pain pathways: A neuro-

anatomical review. Brain Res 1000:40–56, 2004.3. Basbaum AIJ, Thomas M: The Perception of Pain, in Kandel ES, James

H, Jessell Thomas M (eds): Principles of Neural Science, McGraw-HillHealth Professions Division, 2000, pp 472–491.

4. Beniczky S, Tajti J, Timea Varga E, Vecsei L: Evidence-based pharma-cological treatment of neuropathic pain syndromes. J Neural Transm112:735–749, 2005.

5. Bhave G, Gereau RWt: Posttranslational mechanisms of peripheralsensitization. J Neurobiol 61:88–106, 2004.

6. Chattopadhyay M, Wolfe, Darren P, Krisky, David M, Walter, Claire P,Glorioso J, Mata M, Fink D: Gene Therapy of Diabetic NeuropathyUsing HSV-Mediated Transfer of Erythropoietin to Dorsal Root Gan-glion In Vivo Molecular Therapy 11:249, 2005.

7. Clark MR, Treisman GJ: Neurobiology of pain. Adv Psychosom Med25:78–88, 2004.

8. Eaton M: Cell therapy for neuropathic pain in spinal cord injuries.Expert Opin Biol Ther 4:1861–1869, 2004.

9. Eaton MJ, Duplan H: Useful cell lines derived from the adrenal medulla.Mol Cell Endocrinol 228:39–52, 2004.

10. Eaton MJ, Herman JP, Jullien N, Lopez TL, Martinez M, Huang J:Immortalized chromaffin cells disimmortalized with Cre/lox site-di-rected recombination for use in cell therapy for pain after partial nerveinjury. Exp Neurol 175:49–60, 2002.

11. Finegold AA, Mannes AJ, Iadarola MJ: A paracrine paradigm for in vivogene therapy in the central nervous system: treatment of chronic pain.Hum Gene Ther 10:1251–1257, 1999.

12. Fink D, Mata M, Glorioso JC: Cell and gene therapy in the treatment ofpain. Adv Drug Deliv Rev 55:1055–1064, 2003.

13. Frankland PW, Teixeira CM: A pain in the ACC. Mol Pain 1:14, 2005.14. Garrity-Moses ME, Liu JK, Boulis NM: Molecular biology and gene

therapy in the treatment of chronic pain. Neurosurg Clin N Am14:419–435, 2003.

15. Goss JM, M; Goins W, Wu H, Glorioso J: Antinociceptive effect of agenomic herpes simplex virus-based vector expressing human proen-kephalin in rat dorsal root ganglion. Gene Therapy 8:551–556, 2001.

16. Gulwadi AG, Hoane MR, Saydoff JA, Frydel BR, Lindner MD: Nodetectable analgesic effects in the formalin test even with one millionbovine adrenal chromaffin cells. Pain 99:263–271, 2002.

17. Hao S, Mata M, Wolfe D, Glorioso JC, Fink DJ: Gene transfer ofglutamic acid decarboxylase reduces neuropathic pain. Ann Neurol57:914–918, 2005.

18. Hofstetter CP, Holmstrom NA, Lilja JA, Schweinhardt P, Hao J,Spenger C, Wiesenfeld-Hallin Z, Kurpad SN, Frisen J, Olson L: Allo-dynia limits the usefulness of intraspinal neural stem cell grafts; directeddifferentiation improves outcome. Nat Neurosci 8:346–353, 2005.

19. Ji RR, Strichartz G: Cell signaling and the genesis of neuropathic pain.Sci STKE 2004:E14, 2004.

20. Jouvenot YF, John R, Tan S, McNamara A, Chavez RA, Gregory P [233]:Gene control as a therapeutic intervention: Zinc-finger protein transcriptionfactors as regulators of the molecular determinants of neuropathic painMolecular Therapy 9:90, 2004.

21. Jozkowicz A, Dulak J: Helper-dependent adenoviral vectors in experi-mental gene therapy. Acta Biochim Pol 52:589–599, 2005.

22. Klein JP, Tendi EA, Dib-Hajj SD, Fields RD, Waxman SG: Patternedelectrical activity modulates sodium channel expression in sensoryneurons. J Neurosci Res 74:192–198, 2003.

23. Klein S, Svendsen CN: Stem cells in the injured spinal cord: Reducingthe pain and increasing the gain. Nat Neurosci 8:259–260, 2005.

24. Lazorthes Y, Sagen J, Sallerin B, Tkaczuk J, Duplan H, Sol JC, Tafani M,Bes JC: Human chromaffin cell graft into the CSF for cancer pain manage-ment: A prospective phase II clinical study. Pain 87:19–32, 2000.

25. Lin CRW, Chien-Ming L, Ming-Wei C, Jiin T: Intrathecal electropora-tion of doxycycline regulated proopiomelanocortin gene for the neuro-pathic pain therapy in CCI rats. Molecular Therapy 9:pp.241, 2004.

26. Lin CRW, Haibin W, Clifford J, Amaya F: RNA interference targetingEP4 prostanoid receptor attenuates inflammatory pain in rats MolecularTherapy 11:249, 2005.

27. Lin CR, Yang LC, Lee TH, Lee CT, Huang HT, Sun WZ, Cheng JT:Electroporation-mediated pain-killer gene therapy for mononeuropathicrats. Gene Ther 9:1247–1253, 2002.

28. Lindner MD, Francis JM, Plone MA, McDermott PE, Frydel BR, EmerichDF, Saydoff JA: The analgesic potential of intraventricular polymer-encap-sulated adrenal chromaffin cells in a rodent model of chronic neuropathicpain. Exp Clin Psychopharmacol 8:524–538, 2000.

29. Lindner MD, Francis JM, Saydoff JA: Intrathecal polymer-encapsulatedbovine adrenal chromaffin cells fail to produce analgesic effects in thehotplate and formalin test. Exp Neurol 165:370–383, 2000.

30. Lindner MD, Frydel BR, Francis JM, Cain CK: Analgesic effects ofadrenal chromaffin allografts: Contingent on special procedures or dueto experimenter bias? J Pain 4:64–73, 2003.

31. Medina-Kauwe LK, Xie J, Hamm-Alvarez S: Intracellular trafficking ofnonviral vectors. Gene Ther, 2005.

32. Merighi A, Carmignoto G, Gobbo S, Lossi L, Salio C, Vergnano AM,Zonta M: Neurotrophins in spinal cord nociceptive pathways. ProgBrain Res 146:291–321, 2004.

33. Milligan ED, Langer SJ, Sloane EM, He L, Wieseler-Frank J, O’ConnorK, Martin D, Forsayeth JR, Maier SF, Johnson K, Chavez RA, LeinwandLA, Watkins LR: Controlling pathological pain by adenovirally drivenspinal production of the anti-inflammatory cytokine, interleukin-10. EurJ Neurosci 21:2136–2148, 2005.

34. Milligan ED, Melissa J, Langer SJ, Hughes T, Sloane EM, Mahoney J,Jekich B, Cruz PE, Flotte TR, Maier SF, Johnson KW, Leinwand LA,Chavez RA, Watkins LR: Gene delivery for chronic pain control: Mi-crcroencapsulated plasmid DNA encoding the anti-inflammatory cytokinegene, interleukin-10 (IL-10). Molecular Therapy 11:249–250, 2005.

35. Muscoli C, Mollace V, Wheatley J, Masini E, Ndengele M, Wang ZQ,Salvemini D: Superoxide-mediated nitration of spinal manganese super-oxide dismutase: A novel pathway in N-methyl-D-aspartate-mediatedhyperalgesia. Pain 111:96–103, 2004.

36. Ohara PT, Vit JP, Jasmin L: Cortical modulation of pain. Cell Mol LifeSci 62:44–52, 2005.

37. Phillips WJ, Currier BL: Analgesic pharmacology: I. Neurophysiology.J Am Acad Orthop Surg 12:213–220, 2004.

38. Phillips WJ, Currier BL: Analgesic pharmacology: II. Specific analge-sics. J Am Acad Orthop Surg 12:221–233, 2004.

39. Pohl M, Meunier A, Hamon M, Braz J: Gene therapy of chronic pain.Curr Gene Ther 3:223–238, 2003.



40. Price DD: Central neural mechanisms that interrelate sensory andaffective dimensions of pain. Mol Interv 2:392–403, 339, 2002.

41. Przewlocki R, Przewlocka B: Opioids in chronic pain. Eur J Pharmacol429:79–91, 2001.

42. Renn CL, Dorsey SG: The physiology and processing of pain: A review.AACN Clin Issues 16:277–290, 2005.

43. Romanelli P, Esposito V: The functional anatomy of neuropathic pain.Neurosurg Clin N Am 15:257–268, 2004.

44. Sagen J: Chromaffin cell transplants for alleviation of chronic pain.Asaio J 38:24–28, 1992.

45. Sloane EM, Milligan ED, Mahoney J, Jekich B, Levkoff L, Wieseler-Frank J, Maier SF, Forsayeth SF, Leinwand L, Chavez RA, Watkins LR:Nonviral vectors (NVV) encoding the anti-inflammatory cytokine Inter-leukin - 10 (IL10) control chronic constriction injury (CCI) allodynialong term following a variable two injection schedule. Society forNeuroscience, Program 523.524, 2004.

46. Tan SM, Jouvenot Y, Krisky D, Wolfe D, Compos B, et. al.: Zinc fingerprotein transcription factors as potential therapeutic agents for thetreatment of neuropathic pain Molecular Therapy 11:251, 2005.

47. Vanegas H, Schaible HG: Descending control of persistent pain: Inhib-itory or facilitatory? Brain Res Brain Res Rev 46:295–309, 2004.

48. Wang ZQ, Porreca F, Cuzzocrea S, Galen K, Lightfoot R, Masini E,Muscoli C, Mollace V, Ndengele M, Ischiropoulos H, Salvemini D: A

newly identified role for superoxide in inflammatory pain. J PharmacolExp Ther 309:869–878, 2004.

49. Willis WD, Westlund KN: Neuroanatomy of the pain system and of thepathways that modulate pain. J Clin Neurophysiol 14:2–31, 1997.

50. Wilson SY, Bender M, Lu Y, Goins W, Glorioso J: Antihyperalgesiceffects of infection with a preproenkephalin-encoding herpes virus. ProcNat Acad Sci USA 96:3211–3216, 1999.

51. Winn SR, Emerich DF: Managing chronic pain with encapsulated cellimplants releasing catecholamines and endogenous opiods. Front Biosci10:367–378, 2005.

52. Wolfe DH, Hu J, Mata M, Glorioso J, Fink D: A Novel Herpes SimplexVirus Vector Expressing Endomorphin-2: Characterization and Antino-ciceptive Properties. Mol Ther 11:248, 2005.

53. Yao MZ, Gu JF, Wang JH, Sun LY, Liu H, Liu XY: Adenovirus-mediated interleukin-2 gene therapy of nociception. Gene Ther 10:1392–1399, 2003.

54. Yao MZ, Wang JH, Gu JF, Sun LY, Liu H, Zhao ZQ, Liu XY:Interleukin-2 gene has superior antinociceptive effects when deliveredintrathecally. Neuroreport 13:791–794, 2002.

55. Yeomans DC, Peters MC, Trzabazis AZ, Wilson SP, Gilly WF, Ange-lotti T: Decrease in inflammatory hyperalgesia by herpes vector -mediated knock-down of NaV1.7 sodium channels in primary afferents.Society for Neuroscience, Program 62.12, 2004.



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Molecular Mechanism of Pain