Annemarie Ledeboer

Minocycline attenuates mechanical allodynia and proinflammatory cytokine expression in rat models of pain facilitation

Activated glial cells (microglia and astroglia) in the spinal cord play a major role in mediating enhanced pain states by releasing proinflammatory cytokines and other substances thought to facilitate pain transmission. In the present study, we report that intrathecal administration of minocycline, a selective inhibitor of microglial cell activation, inhibits low threshold mechanical allodynia, as measured by the von Frey test, in two models of pain facilitation. In a rat model of neuropathic pain induced by sciatic nerve inflammation (sciatic inflammatory neuropathy, SIN), minocycline delayed the induction of allodynia in both acute and persistent paradigms. Moreover, minocycline was able to attenuate established SIN‐induced allodynia 1 day, but not 1 week later, suggesting a limited role of microglial activation in more perseverative pain states. Our data are consistent with a crucial role for microglial cells in initiating, rather than maintaining, enhanced pain responses. In a model of spinal immune activation by intrathecal HIV‐1 gp120, we show that the anti‐allodynic effects of minocycline are associated with decreased microglial activation, attenuated mRNA expression of interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), IL‐1β‐converting enzyme, TNF‐α‐converting enzyme, IL‐1 receptor antagonist and IL‐10 in lumbar dorsal spinal cord, and reduced IL‐1β and TNF‐α levels in the CSF. In contrast, no significant effects of minocycline were observed on gp120‐induced IL‐6 and cyclooxygenase‐2 expression in spinal cord or CSF IL‐6 levels. Taken together these data highlight the importance of microglial activation in the development of exaggerated pain states.

Interleukin-10, interleukin-4, and transforming growth factor-β differentially regulate lipopolysaccharide-induced production of pro-inflammatory cytokines and nitric oxide in co-cultures of rat astroglial and microglial cells

The pro‐inflammatory cytokines interleukin‐1β (IL‐1β), IL‐6, tumor necrosis factor‐α (TNF‐α), and nitric oxide (NO) can be produced by activated glial cells and play a critical role in various neurological diseases. Using primary co‐cultures of rat microglial and astroglial cells, we investigated the effects of the anti‐inflammatory cytokines transforming growth factor‐β1 (TGF‐β1)/β2, IL‐4, and IL‐10 on the production of (pro‐) inflammatory mediators after stimulation of the cells with lipopolysaccharide (LPS; 0.1 μg/ml, 24 h). IL‐10 (10 and 100 ng/ml) and IL‐4 (5 and 50 U/ml) suppressed the LPS‐induced production of NO, IL‐6, and TNF‐α in a dose‐dependent manner, whereas TGF‐β1/β2 (2 and 20 ng/ml) only suppressed NO production. LPS‐induced levels of IL‐1β were suppressed by IL‐10, but not by IL‐4 and TGF‐β1/β2. Conversely, co‐incubation of the glial cells with LPS and antibodies to TGF‐β1/β2 selectively enhanced LPS‐induced NO production, whereas co‐incubation with antibody to IL‐10 enhanced LPS‐induced production of all pro‐inflammatory cytokines and NO. This finding strongly suggests that effective concentrations of TGF‐β1/β2 and IL‐10 are produced by LPS‐stimulated glial cell co‐cultures. Production of IL‐10 in these co‐cultures was confirmed by measurement of rat IL‐10 by radioimmunoassay. We conclude that anti‐inflammatory cytokines affect the production of inflammatory mediators in LPS‐activated co‐cultures of microglial and astroglial cells differentially. GLIA 30:134–142, 2000.

Involvement of spinal cord nuclear factor κB activation in rat models of proinflammatory cytokine-mediated pain facilitation

Proinflammatory cytokines, such as interleukin‐1β and tumour necrosis factor‐α, are released by activated glial cells in the spinal cord and play a major role in pain facilitation. These cytokines exert their actions, at least partially, through the activation of the transcription factor, nuclear factor κB (NF‐κB). In turn, NF‐κB regulates the transcription of many inflammatory mediators, including cytokines. We have previously shown that intrathecal injection of the human immunodeficiency virus‐1 (HIV‐1) envelope glycoprotein, gp120, induces mechanical allodynia via the release of proinflammatory cytokines. Here, we investigated whether NF‐κB is involved in gp120‐induced pain behaviour in Sprague–Dawley rats. Intrathecal administration of NF‐κB inhibitors, pyrrolidinedithiocarbamate (PDTC) and SN50, prior to gp120 partially attenuated gp120‐induced allodynia. In addition, PDTC delayed and reversed allodynia in a model of neuropathic pain induced by sciatic nerve inflammation. These observations suggest that intrathecal gp120 may lead to activation of NF‐κB within the spinal cord. To reveal NF‐κB activation, we assessed inhibitory factor κBα (IκBα) mRNA expression by in situ hybridization, as NF‐κB activation up‐regulates IκBα gene expression as part of an autoregulatory feedback loop. No or low levels of IκBα mRNA were detected in the lumbar spinal cord of vehicle‐injected rats, whereas IκBα mRNA expression was markedly induced in the spinal cord following intrathecal gp120 in predominantly astrocytes and endothelial cells. Moreover, IκBα mRNA expression positively correlated with proinflammatory cytokine protein levels in lumbosacral cerebrospinal fluid. Together, these results demonstrate that spinal cord NF‐κB activation is involved, at least in part, in exaggerated pain states.

Ibudilast (AV-411): a new class therapeutic candidate for neuropathic pain and opioid withdrawal syndromes

The treatment of neuropathic pain is a major unresolved medical challenge. Present pharmacotherapies only have modest efficacy and numerous side effects. The use of opioid analgesics is additionally coupled with dependence and withdrawal syndromes. Ibudilast (AV-411) is a non-selective phosphodiesterase inhibitor that is also known to suppress glial cell activation. It has been used clinically for other indications with a good safety profile. As glial cell activation is considered to crucially contribute to neuropathic pain as well as opioid dependence and withdrawal, the authors conceived that ibudilast may be useful for treating these conditions. Preclinical data indicate that ibudilast crosses the blood–brain barrier, is well tolerated, is active on oral administration, reduces glial activation and attenuates pain symptoms in diverse rat models of neuropathic pain. In addition, it enhances acute morphine analgesia and attenuates morphine tolerance and withdrawal. Thus ibudilast may improve opioid efficacy and is a promising therapeutic candidate for neuropathic pain, with a novel mechanism of action.

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.

Anti-inflammatory cytokine gene therapy decreases sensory and motor dysfunction in experimental Multiple Sclerosis: MOG-EAE behavioral and anatomical symptom treatment with cytokine gene therapy

Multiple Sclerosis (MS) is an autoimmune inflammatory disease that presents clinically with a range of symptoms including motor, sensory, and cognitive dysfunction as well as demyelination and lesion formation in brain and spinal cord. A variety of animal models of MS have been developed that share many of the pathological hallmarks of MS including motor deficits (ascending paralysis), demyelination and axonal damage of central nervous system (CNS) tissue. In recent years, neuropathic pain has been recognized as a prevalent symptom of MS in a majority of patients. To date, there have been very few investigations into sensory disturbances in animal models of MS. The current work contains the first assessment of hind paw mechanical allodynia (von Frey test) over the course of a relapsing-remitting myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (MOG-EAE) rat model of MS and establishes the utility of this model in examining autoimmune induced sensory dysfunction. We demonstrate periods of both decreased responsiveness to touch that precedes the onset of hind limb paralysis, and increased responsiveness (allodynia) that occurs during the period of motor deficit amelioration traditionally referred to as symptom remission. Furthermore, we tested the ability of our recently characterized anti-inflammatory IL-10 gene therapy to treat the autoimmune inflammation induced behavioral symptoms and tissue histopathological changes. This therapy is shown here to reverse inflammation induced paralysis, to reduce disease associated reduction in sensitivity to touch, to prevent the onset of allodynia, to reverse disease associated loss of body weight, and to suppress CNS glial activation associated with disease progression in this model.

A role for G protein‐coupled receptor kinase 2 in mechanical allodynia

Inflammation and nerve injury can both induce mechanical allodynia via mechanisms involving the production of pro‐inflammatory cytokines and increased neuronal activity. Many neurotransmitters involved in pain signal via G protein‐coupled receptors (GPCRs). GPCR kinase (GRK)2 is a member of the GRK family that regulates agonist‐induced desensitization and signalling of GPCRs. Low intracellular GRK2 levels are associated with increased receptor signalling. The aim of this study was to investigate whether mechanical allodynia is associated with decreased spinal cord GRK2 expression and whether reduced GRK2 increases inflammation‐induced mechanical allodynia. Mechanical allodynia was induced in rats by chronic constriction injury of the sciatic nerve. After 2 weeks, neuronal GRK2 expression was decreased bilaterally in the superficial layers of the lumbar spinal cord dorsal horn. Moreover, interleukin‐1β significantly reduced GRK2 expression ex vivo in spinal cord slices. To investigate whether reduced GRK2 potentiates inflammation‐induced mechanical allodynia, we used GRK2+/– animals expressing decreased GRK2. At baseline, the threshold for mechanical stimulation did not differ between GRK2+/– and wild‐type mice. However, GRK2+/– animals were more sensitive to mechanical stimulation than wild‐type animals after intraplantar λ‐carrageenan injection. We propose cytokine‐induced down‐regulation of spinal cord neuronal GRK2 expression as a novel mechanism that contributes to increased neuronal signalling in mechanical allodynia.

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.