John H. Mahoney

Neonatal Infection Induces Memory Impairments Following an Immune Challenge in Adulthood

Exposure to infectious agents during early postnatal life often alters glucocorticoid responses to stress and immune outcomes in adulthood. The authors examined whether neonatal infection results in memory impairments in adult animals. Rats infected with Escherichia coli (E. coli) as neonates displayed impaired memory for a recently explored context in adulthood. This impairment, however, was only observed in rats that received a peripheral immune challenge (lipopolysaccharide; LPS) immediately following context exposure. Adult rats treated neonatally with E. coli also had decreased hippocampal astrocytes compared with phosphate-buffered saline-treated rats, but displayed increased astrocyte reactivity in the hippocampus and decreased brain interleukin-1beta following lipopolysaccharide. Infection during development appears to alter glia within the hippocampus, which may contribute to altered cytokine responses and memory impairment.

Repeated intrathecal injections of plasmid DNA encoding interleukin-10 produce prolonged reversal of neuropathic pain

&NA; 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 (˜2–3 h), given its short half‐life. Intrathecal gene therapy using viruses encoding IL‐10 can also resolve neuropathic pain, but for only ˜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.

The glial modulatory drug AV411 attenuates mechanical allodynia in rat models of neuropathic pain

Controlling neuropathic pain is an unmet medical need and we set out to identify new therapeutic candidates. AV411 (ibudilast) is a relatively nonselective phosphodiesterase inhibitor that also suppresses glial-cell activation and can partition into the CNS. Recent data strongly implicate activated glial cells in the spinal cord in the development and maintenance of neuropathic pain. We hypothesized that AV411 might be effective in the treatment of neuropathic pain and, hence, tested whether it attenuates the mechanical allodynia induced in rats by chronic constriction injury (CCI) of the sciatic nerve, spinal nerve ligation (SNL) and the chemotherapeutic paclitaxel (Taxol). Twice-daily systemic administration of AV411 for multiple days resulted in a sustained attenuation of CCI-induced allodynia. Reversal of allodynia was of similar magnitude to that observed with gabapentin and enhanced efficacy was observed in combination. We further show that multi-day AV411 reduces SNL-induced allodynia, and reverses and prevents paclitaxel-induced allodynia. Also, AV411 cotreatment attenuates tolerance to morphine in nerve-injured rats. Safety pharmacology, pharmacokinetic and initial mechanistic analyses were also performed. Overall, the results indicate that AV411 is effective in diverse models of neuropathic pain and support further exploration of its potential as a therapeutic agent for the treatment of neuropathic pain.

Intrathecal polymer-based interleukin-10 gene delivery for neuropathic pain

Research on communication between glia and neurons has increased in the past decade. The onset of neuropathic pain, a major clinical problem that is not resolved by available therapeutics, involves activation of spinal cord glia through the release of proinflammatory cytokines in acute animal models of neuropathic pain. Here, we demonstrate for the first time that the spinal action of the proinflammatory cytokine, interleukin 1 (IL-1) is involved in maintaining persistent (2 months) allodynia induced by chronic-constriction injury (CCI). The anti-inflammatory cytokine IL-10 can suppress proinflammatory cytokines and spinal cord glial amplification of pain. Given that IL-1 is a key mediator of neuropathic pain, developing a clinically viable means of long-term delivery of IL-10 to the spinal cord is desirable. High doses of intrathecal IL-10-gene therapy using naked plasmid DNA (free pDNA-IL-10) is effective, but the dose required limits its potential clinical utility. Here we show that intrathecal gene therapy for neuropathic pain is improved sufficiently using two, distinct synthetic polymers, poly(lactic-co-glycolic) and polyethylenimine, that substantially lower doses of pDNA-IL-10 are effective. In conclusion, synthetic polymers used as i.t. gene-delivery systems are well-tolerated and improve the long-duration efficacy of pDNA-IL-10 gene therapy.

A novel immune-to-CNS communication pathway: cells of the meninges surrounding the spinal cord CSF space produce proinflammatory cytokines in response to an inflammatory stimulus.

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.

Immunological priming potentiates non-viral anti-inflammatory gene therapy treatment of neuropathic pain

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-10F129S 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.

Novel approaches to control paclitaxel-induced neuropathic pain: Efficacy of interleukin-10 gene therapy and the blood–brain barrier permeable glial modulator, AV411

(DPC). Levels of inflammatory cytokines, TNF-a and IL6, were quite low or below the detection limits of the assays. This is related to the fact that these were healthy young monkeys with no observable infectious or inflammatory process. Post test cortisol levels averaged 52.5 ± 20.7 mg/ dl, range 12.5–120, for the entire cohort. Plasma cortisol was unrelated to any of the measures of early maternal experience but was related to agitation behaviors during the novelty test. In contrast c-INF showed consistent and significant relationships to early maternal experience. The young infants that had less contact with the mother in the second and third month of life had significantly higher plasma c-INF levels two years later as juveniles (ps < .001). The high c-INF juveniles had relatively neglectful mothers who were significantly more likely to leave their infants and less likely to restrain them. As infants, they had significantly higher rates of both approaching and leaving the mothers, suggesting that they took a more active role in exploring the environment at an early age. The extent to which these differences contribute to later health outcomes is not known at this time. These animals are part of a longitudinal study of a number of disease processes.

644. Gene Delivery for Chronic Pain Control: Micrcroencapsulated Plasmid DNA Encoding the Anti-Inflammatory Cytokine Gene, Interleukin-10 (IL-10)

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.