Glial−neuronal interactions are crucial processes in neuromodulation and synaptic plasticity. The neuregulin 1 family of growth and differentiation factors have been implicated as bidirectional signaling molecules that are involved in mediating some of these interactions. We have shown previously that neuregulin 1 expression is regulated by the gonadal hormones progesterone and 17β-estradiol in the CNS, which might represent a novel, indirect mechanism of the neuromodulatory actions of these gonadal hormones. In the present study, we sought to determine the effects of progesterone and 17β-estradiol on neuregulin 1 expression in rat cortical astrocytes and neurons in vitro. We observed that progesterone increased the expression of neuregulin 1 mRNA and protein in a dose-dependent manner in cultured astrocytes, which was blocked by the progesterone receptor antagonist RU-486. In contrast, 17β-estradiol did not increase either neuregulin 1 mRNA or protein in astrocytes. We observed no effect of either progesterone or 17β-estradiol on neuregulin 1 mRNA and protein in rat cortical neurons in vitro. Finally, we observed that treatment of cortical neurons with recombinant NRG1-β1 caused PSD-95 to localize in puncta similar to that observed following treatment with astrocyte-conditioned medium. These results demonstrate that progesterone regulates neuregulin 1 expression, principally in astrocytes. This might represent a novel mechanism of progesterone-mediated modulation of neurotransmission through the regulation of astrocyte-derived neuregulin 1.

The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling and inflammation. Mice homozygous for a null allele at the SHP-1 locus have a phenotype of severe inflammation and are hyper-responsive to the TLR4 ligand LPS. TLR4 stimulation in the CNS has been linked to both neuropathic pain and sickness behaviors. To determine if reduction in SHP-1 expression affects LPS-induced behaviors, responses of heterozygous SHP-1-deficient (me/+) and wild-type (+/+) mice to LPS were measured. Chronic (4-week) treatment with LPS induced avoidant behaviors indicative of fear/anxiety in me/+, but not +/+, mice. These behaviors were correlated with a LPS-induced type 2 cytokine, cytokine receptor, and immune effector arginase profile in the brains of me/+ mice not found in +/+ mice. Me/+ mice also had a constitutively greater level of TLR4 in the CNS than +/+ mice. Additionally, me/+ mice displayed constitutively increased thermal sensitivity compared to +/+ mice, measured by the tail-flick test. Moreover, me/+ glial cultures were more responsive to LPS than +/+ glia. Therefore, the reduced expression of SHP-1 in me/+ imparts haploinsufficiency with respect to the control of CNS TLR4 and pain signaling. Furthermore, type 2 cytokines become prevalent during chronic TLR4 hyperstimulation in the CNS and are associated positively with behaviors that are usually linked to type 1 pro-inflammatory cytokines. These findings question the notion that type 2 immunity is solely anti-inflammatory in the CNS and indicate that type 2 immunity induces/potentiates CNS inflammatory processes.

Satellite glial cells (SGCs) tightly envelop the perikarya of primary sensory neurons in peripheral ganglion and are identified by their morphology and the presence of proteins not found in ganglion neurons. These SGC-unique proteins include the inwardly rectifying K+ channel Kir4.1, the connexin-43 (Cx43) subunit of gap junctions, the purinergic receptor P2Y4 and soluble guanylate cyclase. We also present evidence that the small-conductance Ca2+-activated K+ channel SK3 is present only in SGCs and that SGCs divide following nerve injury. All the above proteins are involved, either directly or indirectly, in potassium ion (K+) buffering and, thus, can influence the level of neuronal excitability, which, in turn, has been associated with neuropathic pain conditions. We used in vivo RNA interference to reduce the expression of Cx43 (present only in SGCs) in the rat trigeminal ganglion and show that this results in the development of spontaneous pain behavior. The pain behavior is present only when Cx43 is reduced and returns to normal when Cx43 concentrations are restored. This finding shows that perturbation of a single SGC-specific protein is sufficient to induce pain responses and demonstrates the importance of PNS glial cell activity in the pathophysiology of neuropathic pain.

Although pain is regarded traditionally as neuronally mediated, recent progress shows an important role of spinal glial cells in persistent pain sensitization. Mounting evidence has implicated spinal microglia in the development of chronic pain (e.g. neuropathic pain after peripheral nerve injury). Less is known about the role of astrocytes in pain regulation. However, astrocytes have very close contact with synapses and maintain homeostasis in the extracellular environment. In this review, we provide evidence to support a role of spinal astrocytes in maintaining chronic pain. In particular, c-Jun N-terminal kinase (JNK) is activated persistently in spinal astrocytes in a neuropathic pain condition produced by spinal nerve ligation. This activation is required for the maintenance of neuropathic pain because spinal infusion of JNK inhibitors can reverse mechanical allodynia, a major symptom of neuropathic pain. Further study reveals that JNK is activated strongly in astrocytes by basic fibroblast growth factor (bFGF), an astroglial activator. Intrathecal infusion of bFGF also produces persistent mechanical allodynia. After peripheral nerve injury, bFGF might be produced by primary sensory neurons and spinal astrocytes because nerve injury produces robust bFGF upregulation in both cell types. Therefore, the bFGF/JNK pathway is an important signalling pathway in spinal astrocytes for chronic pain sensitization. Investigation of signaling mechanisms in spinal astrocytes will identify new molecular targets for the management of chronic pain.

Given the growing body of evidence for a role of glia in pain modulation, it is plausible that the exaggerated visceral pain in chronic conditions might be regulated by glial activation. In this study, we have investigated a possible role for microglia in rats with chronic visceral hypersensitivity and previously documented altered neuronal function. Experiments were performed on adult male Sprague-Dawley rats pre-treated with neonatal colon irritation (CI) and on control rats. Effects of fractalkine (FKN, a chemokine involved in neuron-to-microglia signaling) and of minocycline (an inhibitor of microglia) on visceral sensitivity were examined. Visceral sensitivity was assessed by recording the electromyographic (EMG) responses to graded colorectal distension (CRD) in mildly sedated rats. Responses to CRD were recorded before and after injection of FKN, minocycline or vehicle. Somatic thermal hyperalgesia was measured by latency of paw withdrawal to radiant heat. The pattern and intensity of microglial distribution at L6–S2 in the spinal cord was also compared in rats with CI and controls by fluorescence microscopy using OX-42. Results show that: (1) FKN significantly facilitated EMG responses to noxious CRD by >52% in control rats. FKN also induced thermal hyperalgesia in control rats, consistent with previous reports; (2) minocycline significantly inhibited EMG responses to noxious CRD by >70% in rats with CI compared to controls 60 min after injection. The anti-nociceptive effect of minocycline lasted for 180 min in rats with CI, reaching peak values 60 min after injection. Our results show that FKN enhances visceral and somatic nociception, whereas minocycline inhibits visceral hypersensitivity in chronically sensitized rats, which indicates a role for microglia in visceral hypersensitivity.

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.

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.

First-generation adenovirus can be engineered with powerful promoters to drive expression of therapeutic transgenes. Numerous clinical trials for glioblastoma multiforme using first generation adenoviral vectors have either been performed or are ongoing, including an ongoing, Phase III, multicenter trial in Europe and Israel (Ark Therapeutics, Inc.). Although in the absence of anti-adenovirus immune responses expression in the brain lasts 6–18 months, systemic infection with adenovirus induces immune responses that inhibit dramatically therapeutic transgene expression from first generation adenoviral vectors, thus, potentially compromising therapeutic efficacy. Here, we show evidence of an immunization threshold for the dose that generates an immune response strong enough to eliminate transgene expression from the CNS. For the systemic immunization to eliminate transgene expression from the brain, ≥1×107 infectious units (iu) of adenovirus need to be used as immunogen. Furthermore, this immune response eliminates >90% of transgene expression from 1×107–1×103 iu of vector injected into the striatum 60 days earlier. Importantly, elimination of transgene expression is independent of the nature of the promoter that drives transgene expression and is accompanied by brain infiltration of CD8+ T cells and macrophages. In conclusion, once the threshold for systemic immunization (i.e. 1×107 iu) is crossed, the immune response eliminates transgene expression by >90% even from brains that receive as little as 1000 iu of adenoviral vectors, independently of the type of promoter that drives expression.