We recently described a non-viral gene therapy paradigm offering long-term resolution of established neuropathic pain in several animal models. Here, the requirements for long-term therapeutic effects are described, and evidence is provided for a mechanism of action based on immunological priming of the intrathecal (i.t.) space. Long-term pain reversal was achieved when two i.t. injections of various naked plasmid DNA doses were separated by 5 h to 3 days. We show that an initial DNA injection, regardless of whether a transgene is included, leads to an accumulation of phagocytic innate immune cells. This accumulation coincides with the time in which subsequent DNA injection efficacy is potentiated. We show the ability of non-coding DNA to induce short-term pain reversal that is dependent on endogenous interleukin-10 (IL-10) signaling. Long-term efficacy requires the inclusion of an IL-10(F129S) transgene in the second injection. Blockade of IL-10, by a neutralizing antibody, either between the two injections or after the second injection induces therapeutic failure. These results show that this gene therapy paradigm uses an initial 'priming' injection of DNA to induce accumulation of phagocytic immune cells, allowing for potentiated efficacy of a subsequent 'therapeutic' DNA injection in a time- and dose-dependent manner.

Spinal proinflammatory cytokines are powerful pain-enhancing signals that contribute to pain following peripheral nerve injury (neuropathic pain). Recently, one proinflammatory cytokine, interleukin-1, was also implicated in the loss of analgesia upon repeated morphine exposure (tolerance). In contrast to prior literature, we demonstrate that the action of several spinal proinflammatory cytokines oppose systemic and intrathecal opioid analgesia, causing reduced pain suppression. In vitro morphine exposure of lumbar dorsal spinal cord caused significant increases in proinflammatory cytokine and chemokine release. Opposition of analgesia by proinflammatory cytokines is rapid, occurring < or =5 min after intrathecal (perispinal) opioid administration. We document that opposition of analgesia by proinflammatory cytokines cannot be accounted for by an alteration in spinal morphine concentrations. The acute anti-analgesic effects of proinflammatory cytokines occur in a p38 mitogen-activated protein kinase and nitric oxide dependent fashion. Chronic intrathecal morphine or methadone significantly increased spinal glial activation (toll-like receptor 4 mRNA and protein) and the expression of multiple chemokines and cytokines, combined with development of analgesic tolerance and pain enhancement (hyperalgesia, allodynia). Statistical analysis demonstrated that a cluster of cytokines and chemokines was linked with pain-related behavioral changes. Moreover, blockade of spinal proinflammatory cytokines during a stringent morphine regimen previously associated with altered neuronal function also attenuated enhanced pain, supportive that proinflammatory cytokines are importantly involved in tolerance induced by such regimens. These data implicate multiple opioid-induced spinal proinflammatory cytokines in opposing both acute and chronic opioid analgesia, and provide a novel mechanism for the opposition of acute opioid analgesia.

Pain is enhanced in response to elevations of proinflammatory cytokines in spinal cerebrospinal fluid (CSF), following either intrathecal injection of these cytokines or intrathecal immune challenge with HIV-1 gp120 that induces cytokine release. Spinal cord glia have been assumed to be the source of endogenous proinflammatory cytokines that enhance pain. However, assuming that spinal cord glia are the sole source of CSF cytokines may be an underestimate, as the cellular composition of the meninges surrounding the spinal cord CSF space includes several cell types known to produce proinflammatory cytokines. The present experiments provide the first investigation of the immunocompetent nature of the spinal cord meninges. Here, we explore whether rat meninges are responsive to intrathecal gp120. These studies demonstrate that: (a) intrathecal gp120 upregulates meningeal gene expression of proinflammatory signals, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin 6 (IL-6), and inducible nitric oxide synthase (iNOS), and (b) intrathecal gp120 induces meningeal release of TNF-alpha, IL-1beta, and IL-6. In addition, stimulation of isolated meninges in vitro with gp120 induced the release of TNF-alpha and IL-1beta, indicating that the resident cells of the meninges are able to respond without immune cell recruitment. Taken together, these data document that the meninges are responsive to immunogenic stimuli in the CSF and that the meninges may be a source of immune products detected in CSF. The ability of the meninges to release to proinflammatory signals suggests a potential role in the modulation of pain.

Neuropathic pain is a major clinical problem unresolved by available therapeutics. Spinal cord glia play a pivotal role in neuropathic pain, via the release of proinflammatory cytokines. Anti-inflammatory cytokines, like interleukin-10 (IL-10), suppress proinflammatory cytokines. Thus, IL-10 may provide a means for controlling glial amplification of pain. We recently documented that intrathecal IL-10 protein resolves neuropathic pain, albeit briefly (approximately 2-3 h), given its short half-life. Intrathecal gene therapy using viruses encoding IL-10 can also resolve neuropathic pain, but for only approximately 2 weeks. Here, we report a novel approach that dramatically increases the efficacy of intrathecal IL-10 gene therapy. Repeated intrathecal delivery of plasmid DNA vectors encoding IL-10 (pDNA-IL-10) abolished neuropathic pain for greater than 40 days. Naked pDNA-IL-10 reversed chronic constriction injury (CCI)-induced allodynia both shortly after nerve injury as well as 2 months later. This supports that spinal proinflammatory cytokines are important in both the initiation and maintenance of neuropathic pain. Importantly, pDNA-IL-10 gene therapy reversed mechanical allodynia induced by CCI, returning rats to normal pain responsiveness, without additional analgesia. Together, these data suggest that intrathecal IL-10 gene therapy may provide a novel approach for prolonged clinical pain control.

Paclitaxel is a commonly used cancer chemotherapy drug that frequently causes painful peripheral neuropathies. The mechanisms underlying this dose-limiting side effect are poorly understood. Growing evidence supports that proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), released by activated spinal glial cells and within the dorsal root ganglia (DRG) are critical in enhancing pain in various animal models of neuropathic pain. Whether these cytokines are involved in paclitaxel-induced neuropathy is unknown. Here, using a rat neuropathic pain model induced by repeated systemic paclitaxel injections, we examined whether paclitaxel upregulates proinflammatory cytokine gene expression, and whether these changes and paclitaxel-induced mechanical allodynia can be attenuated by intrathecal IL-1 receptor antagonist (IL-1ra) or intrathecal delivery of plasmid DNA encoding the anti-inflammatory cytokine, interleukin-10 (IL-10). The data show that paclitaxel treatment induces mRNA expression of IL-1, TNF, and immune cell markers in lumbar DRG. Intrathecal IL-1ra reversed paclitaxel-induced allodynia and intrathecal IL-10 gene therapy both prevented, and progressively reversed, this allodynic state. Moreover, IL-10 gene therapy resulted in increased IL-10 mRNA levels in lumbar DRG and meninges, measured 2 weeks after initiation of therapy, whereas paclitaxel-induced expression of IL-1, TNF, and CD11b mRNA in lumbar DRG was markedly decreased. Taken together, these data support that paclitaxel-induced neuropathic pain is mediated by proinflammatory cytokines, possibly released by activated immune cells in the DRG. We propose that targeting the production of proinflammatory cytokines by intrathecal IL-10 gene therapy may be a promising therapeutic strategy for the relief of paclitaxel-induced neuropathic pain.

Chronic pathological pain is a major unresolved clinical problem. Spinal cord glial cells are important in diverse forms of enhanced pain states via their release of proinflammatory cytokines. Interleukin-10 (IL-10), a potent anti-inflammatory cytokine, suppresses proinflammatory cytokine production & activity. We have shown that spinal delivery (intrathecal; i.t.) of IL-10 protein resolves animal models of pathological pain. Both i.t. IL-10 protein or i.t. viral vector-delivered IL-10 gene lead to transient therapeutic effects (2hr & 2 wk, respectively). We have recently reported that i.t. naked plasmid DNA encoding IL-10 (pDNA-IL-10) leads to a substantial improvement in the duration of therapeutic pain control (40 days). However, transgene IL-10 may be compromised &/or cleared by cell division. Recently, a chromosomal scaffold/matrix- attached region (S/MAR) linked to the simian virus 40 origin of replication was reported to stably propagate episomally in mammalian cells. Mitotic stability occurs via specific interactions with nuclear matrix proteins. We sought determine if S/MAR inserted into our current plasmid (pDNA-S/MARS-IL10) could improve upon the therapeutic duration of pain control in an animal model of neuropathic pain. The model we use is hypersensitivity to light touch [allodynia] produced by chronic constriction injury [CCI] of the sciatic nerve. Naked pDNA-S/MARS-IL10 was injected peri- spinally (intrathecally; i.t.) after CCI-sensitivity was induced. Behavioral measures were assessed in rats prior to & at 3 & 10 days after induction of CCI. Intrathecal injections were given on Day 10 & Day 12 post-CCI consisting of pDNA-S/MARS-IL10 (100 ug/injection in 18 ul), or pDNA-IL-10 (100 ug/injection in 18 ul). A second i.t. pDNA-S/MARS-IL10 (1 or 25 ug/injection in 5 ul), or pDNA-IL10 (25 ug in 5 ul) injection was given 2 days later. Allodynia was reassessed every 4 days following the second DNA injection. Allodynia was stable through day 10 after CCI induction. An immediate & robust improvement of pain reversal was observed within 24 hr after the first injection of pDNA-S/MARS-IL10 vs pDNA-IL-10. Animals have remained reversed from CCI-induced allodynia after the second i.t. pDNA-S/MARS-IL10 and 25 ug is most effective.Studies are ongoing and will be terminated 90 days after CCI or when CCI-induced allodynia returns. Simultaneous studies are determining whether 1) a single i.t. injection of pDNA-S/ MARS-IL10 is sufficient for long-term pain control, 2) transgene expression by protein analysis can be detected at 1, 2 &/or 3 months in cerebrospinal fluid after injection in behaviorally verified rats, and 3) the distribution of transgene expression in superficial spinal cord or deeper parenchymal layers. We thank Dr. Hans J. Lipps for the gift of plasmid containing pDNA-S/MARS. Support: NIH HL56510, DA018156 & DA015642.

Chronic pain control is a major unresolved clinical problem. Spinal cord astrocytes & microglia are critically involved in the creation & maintenance of diverse enhanced pain states via the release of proinflammatory cytokines. Interleukin-10 (IL-10), a potent anti-inflammatory cytokine, suppresses proinflammatory cytokine production & activity. We have previously shown that administration of IL-10 protein directly or via a viral vector encoding it into the spinal cord (intrathecal, i.t.) reverses neuropathic pain in the rat; although direct administration of IL-10 protein showed a very short-term reversal. We sought to determine whether neuropathic pain (hypersensitivity to light touch [allodynia] produced by chronic constriction injury [CCI] of the sciatic nerve) could be reversed by: 1) single or repeated i.t. delivery of naked plasmid DNA encoding rat IL-10 (pIL10), 2) single or repeated i.t. delivery of pIL10 treated with the cationic polymer, polyethyleneimine (PEI- pIL10), and 3) single or repeated i.t. pIL10 encapsulated in micro-particles prepared from FDA-approved biodegradable copolymers of polylactide and polyglycolide (PLGA) (PLGA- pIL10). Lastly, we examined whether i.t. rhodamine-labeled PLGA-micro-particles could be visualized in spinal cord using confocal microscopy in naive rats. Behavioral measures were assessed prior to & at 3 & 10 days post CCI. I.t. injections were given on day 10 post-CCI, consisting of pIL10 (100 ug/injection), control plasmid encoding jellyfish green fluorescent protein (pGFP; 100 ug/injection), vehicle (3% sucrose in phosphate buffered saline), PEI- pIL10 (10 ug/injection) or PLGA- pIL10 (350 ug PGLA/injection). For repeated injections, a 2nd injection was given on day 13. Allodynia was reassessed every 1 to 4 days. Allodynia was stable in control treated rats but was reversed (prolonged reversal lasting 40+ days), in rats given 2 i.t. injections of: 1) pIL10 2) PEI- pIL10 & 3) PLGA-IL10. Single i.t. injections of pIL10, PEI- pIL10 or PLGA- pIL10 produced a brief 3-6 day reversal of allodynia. Importantly, rhodamine-labeled PLGA-micro-particles were visible immediately & at 3 days after i.t. injection. Ongoing studies using i.t. rhodamine-labeled PLGA-micro-particles (either without DNA or with pGFP DNA) are aimed at examining the spread, the distribution in superficial spinal cord or deeper parenchymal layers & gene expression. This approach to pain control represents a dramatic departure from all other available therapies. Support: Avigen & NIH HL56510, DA015656, DA018156 & DA015642.

Controlling chronic pain in humans is a major unresolved problem. Spinal cord astrocytes and microglia are critically involved in the creation and maintenance of diverse enhanced pain states via the release of proinflammatory cytokines. Interluekin-10 (IL-10), a potent anti-inflammatory cytokine, suppresses proinflammatory cytokine production and activity. Control of chronic neuropathic pain such as sensitivity to light touch (allodynia) requires chronic spinal delivery of IL-10. Chronic pain can be surgically induced in rats by loose ligature of chromic cat gut around the sciatic nerve in four places. This creates a chronic constriction injury (CCI) as the nerve bundle swells against the irritating ligature. The CCI model produces an enhanced pain phenotype for 3 months. We have demonstrated that this pain is reversed transiently (4hrs.) through intrathecal injection of recombinant IL-10 protein and that two temporally spaced injections of an IL-10 expression vector containing inverted terminal repeats (ITRs) reverses this pain long term (3+ months, Milligan et al., these proceedings). The role of ITRs in gene expression is not well known. However, there is evidence that they possess transcription promoter and enhancer activity (Flotte et al., 1992 Am J Respir Cell Mol Biol. 7:p349-56 and 1993 J Biol Chem. 268: p3781-90). Based on this and other evidence in the literature we sought to determine if the ITRs in our expression vector, which are located 5' of the CMV enhancer and Chicken â-actin promoter (5' ITR) and 3' of the SV 40 poly A tail sequence (3' ITR), influence reversal of pain in the CCI model. To this end, we deleted both ITRs from the expression vector and found that it was incapable of producing pain reversal. We next asked if one or both ITRs are necessary for pain reversal. We found that plasmids containing just the 3' or 5' ITR were both effective in reversing the chronic pain state. Ongoing studies seek to determine the effect of multimerization of the injected plasmid on pain reversal and gene expression and to further characterize the role of ITRs on gene expression and pain reversal in the CCI model.

Chronic abdominal pain is a common symptom of great clinical significance in several areas of medicine. In many cases no organic cause can be established resulting in the classification as functional gastrointestinal disorder. Irritable Bowel Syndrome (IBS) is the most common of these conditions and is considered an important public health problem because it can be disabling and constitutes a major social and economic burden given the lack of effective treatments. IBS aetiology is most likely multi-factorial involving biological, psychological and social factors. Visceral hyperalgesia (or hypersensitivity) and visceral hypervigilance, which could be mediated by peripheral, spinal, and/or central pathways, constitute key concepts in current research on pathophysiological mechanisms of visceral hyperalgesia. The role of central nervous system mechanisms along the “brain–gut axis” is increasingly appreciated, owing to accumulating evidence from brain imaging studies that neural processing of visceral stimuli is altered in IBS together with long-standing knowledge regarding the contribution of stress and negative emotions to symptom frequency and severity. At the same time, there is also growing evidence suggesting that peripheral immune mechanisms and disturbed neuro-immune communication could play a role in the pathophysiology of visceral hyperalgesia. This review presents recent advances in research on the pathophysiology of visceral hyperalgesia in IBS, with a focus on the role of stress and anxiety in central and peripheral response to visceral pain stimuli. Together, these findings support that in addition to lower pain thresholds displayed by a significant proportion of patients, the evaluation of pain appears to be altered in IBS. This may be attributable to affective disturbances, negative emotions in anticipation of or during visceral stimulation, and altered pain-related expectations and learning processes. Disturbed “top-down” emotional and cognitive pain modulation in IBS is reflected by functional and possibly structural brain changes involving prefrontal as well as cingulate regions. At the same time, there is growing evidence linking peripheral and mucosal immune changes and abdominal pain in IBS, supporting disturbed peripheral pain signalling. Findings in post-infectious IBS emphasize the interaction between centrally-mediated psychosocial risk factors and local inflammation in predicting long-term IBS symptoms. Investigating afferent immune-to-brain communication in visceral hyperalgesia as a component of the sickness response constitutes a promising future research goal.

Glia are now recognized as important contributors in pathological pain creation and maintenance. Spinal cord glia exhibit extensive gap junctional connectivity, raising the possibility that glia are involved in the contralateral spread of excitation resulting in mirror image pain. In the present experiments, the gap junction decoupler carbenoxolone was administered intrathecally after induction of neuropathic pain in response to sciatic nerve inflammation (sciatic inflammatory neuropathy) or partial nerve injury (chronic constriction injury). In both neuropathic pain models, a low dose of carbenoxolone reversed mirror image mechanical allodynia, while leaving ipsilateral mechanical allodynia unaffected. Ipsilateral thermal hyperalgesia was briefly attenuated. Critically, blockade of mechanical allodynia and thermal hyperalgesia was not observed in response to intrathecal glycyrrhizic acid, a compound similar to carbenoxolone in all respects but it does not decouple gap junctions. Thus, blockade of mechanical allodynia and thermal hyperalgesia by carbenoxolone does appear to reflect an effect on gap junctions. Examination of carbenoxolone's effects on intrathecal human immunodeficiency virus type 1 gp120 showed that blockade of pain facilitation might result, at least in part, via suppression of interleukin-1 and, in turn, interleukin-6. These data provide the first suggestion that spread of excitation via gap junctions might contribute importantly to inflammatory and traumatic neuropathic pain.Perspective: The current studies provide evidence for involvement of gap junctions in spinal cord pain facilitation. Intrathecal carbenoxolone, a gap junction decoupler, reversed neuropathy-induced mirror image pain and intrathecal gp120-induced allodynia. in addition, it decreased gp120-induced proinflammatory cytokines. This suggests gap junction activation might lead to proinflammatory cytokine release by distantly activated glia. (C) 2004 by the American Pain Society.