Joyce A. De Leo

The tetrapartite synapse : Path to CNS sensitization and chronic pain

A recent plethora of studies has characterized a central nervous system (CNS) neuroimmune response in animal models of persistent pain. This response, which may be the driving force for neuronal hypersensitivity in chronic pain states, involves ‘‘activation’’ of spinal and supraspinal glial cells, which when stimulated may increase production of a host of inflammatory/algesic mediators, such as cytokines and chemokines. Unclear, however, is how this neuroimmune activation can actually cause the downstream electrophysiological mechanisms of enhanced neuronal firing or decreased thresholds to firing; and therefore, produce heightened responses to noxious and non-noxious stimuli. The contribution of CNS proinflammatory cytokines to neuronal sensitization is unclear. For example, the use of specific cytokine inhibitors or the administration of an anti-inflammatory cytokine for the treatment of chronic pain has lacked definitive mechanisms and/or effective outcomes. Many of these reports use terms such as ‘‘activation’’ and ‘‘glia’’, without clarity or precision. The heterogeneity of glia and the intricacies of glial function necessitate a reexamination of the terminology in the pain field. Glia, including astrocytes and microglia, rather than neurons, are now the focus in studies of the regulation of synaptic strength and plasticity and the actual generation of central sensitization. In this paper we describe a tetrapartite synapse, which includes an astrocyte, a

Current Review of in Vivo GBM Rodent Models: Emphasis on the CNS-1 Tumour Model

GBM (glioblastoma multiforme) is a highly aggressive brain tumour with very poor prognosis despite multi-modalities of treatment. Furthermore, recent failure of targeted therapy for these tumours highlights the need of appropriate rodent models for preclinical studies. In this review, we highlight the most commonly used rodent models (U251, U86, GL261, C6, 9L and CNS-1) with a focus on the pathological and genetic similarities to the human disease. We end with a comprehensive review of the CNS-1 rodent model.

Inhibition of microglial P2X4 receptors attenuates morphine tolerance, Iba1, GFAP and μ opioid receptor protein expression while enhancing perivascular microglial ED2

&NA; Anti‐nociceptive tolerance to opioids is a well‐described phenomenon, which severely limits the clinical efficacy of opioids for the treatment of chronic pain syndromes. The mechanisms that drive anti‐nociceptive tolerance, however, are less well understood. We have previously shown that glia have a central role in the development of morphine tolerance and that administration of a glial modulating agent attenuated tolerance formation. Recently, we have demonstrated that morphine enhances microglial Iba1 expression and P2X4 receptor‐mediated microglial migration via direct &mgr; opioid receptor signaling in in vitro microglial cultures. We hypothesize that P2X4 receptors drive morphine tolerance and modulate morphine‐induced spinal glial reactivity. Additionally, we hypothesize that perivascular microglia play a role in morphine tolerance and that P2X4 receptor expression regulates perivascular microglia ED2 expression. To test these hypotheses, rats were implanted with osmotic minipumps releasing morphine or saline subcutaneously for seven days. Beginning three days prior to morphine treatment, P2X4 receptor antisense oligonucleotide (asODN) was injected intrathecally daily, to selectively inhibit P2X4 receptor expression. P2X4 receptor asODN treatment inhibited morphine‐induced P2X4 receptor expression and blocked anti‐nociceptive tolerance to systemically administered morphine. P2X4 receptor asODN treatment also attenuated the morphine‐dependent increase of spinal ionized calcium binding protein (Iba1), glial fibrillary acidic protein (GFAP) and &mgr; opioid receptor protein expression. Chronic morphine also decreased perivascular microglial ED2 expression, which was reversed by P2X4 receptor asODN. Together, these data suggest that the modulation of P2X4 receptor expression on microglia and perivascular microglia may prove an attractive target for adjuvant therapy to attenuate opioid‐induced anti‐nociceptive tolerance.

Propentofylline: Glial Modulation, Neuroprotection, and Alleviation of Chronic Pain

Propentofylline is a unique methylxanthine with clear cyclic AMP, phosphodiesterase, and adenosine actions, including enhanced synaptic adenosine signaling. Both in vitro and in vivo studies have demonstrated profound neuroprotective, antiproliferative, and anti-inflammatory effects of propentofylline. Propentofylline has shown efficacy in preclinical models of stroke, opioid tolerance, and acute and chronic pain. Clinically, propentofylline has shown efficacy in degenerative and vascular dementia, and as a potential adjuvant treatment for schizophrenia and multiple sclerosis. Possible mechanisms of action include a direct glial modulation to decrease a reactive phenotype, decrease glial production and release of damaging proinflammatory factors, and enhancement of astrocyte-mediated glutamate clearance. This chapter reviews the literature that supports a myriad of protective actions of this small molecule and implicates propentofylline as a potential therapeutic for the treatment of chronic pain. From these studies, we propose a CNS multipartite synaptic action of propentofylline that includes modulation of pre- and postsynaptic neurons, astrocytes, and microglia in the treatment of chronic pain syndromes, including, but not limited to, neuropathic pain.

Propentofylline decreases tumor growth in a rodent model of glioblastoma multiforme by a direct mechanism on microglia.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer, with a median survival of less than 2 years after diagnosis. The tumor microenvironment plays a critical role in tumor invasion and progression. Microglia and infiltrating macrophages are the most abundant immune cells in the tumor. In the present study, we demonstrate that systemic propentofylline (PPF), an atypical methylxanthine with central nervous system (CNS) glial modulating and anti-inflammatory actions, significantly decreased tumor growth in a CNS-1 rat model of GBM by targeting microglia and not tumor cells. Rats received tumor injections of 1 × 10(5) CNS-1 cells in the right striatum with daily intraperitonial injections of PPF (50 mg/kg) or saline beginning the day of tumor injection. PPF did not cause apoptosis or decrease proliferation of CNS-1 tumor cells. Furthermore, we demonstrate, using in vitro methods, that PPF decreased microglial migration toward CNS-1 tumor cells and decreased MMP-9 expression. The effects of PPF were shown to be specific to microglia and not peripheral macrophages. These results support a differential functional role of resident microglia and infiltrating macrophages in the brain tumor environment. Our data highlight microglia as a crucial target for future therapeutic development and present PPF as a possible drug for treatment of human GBM.

Critical role of microglial CD40 in the maintenance of mechanical hypersensitivity in a murine model of neuropathic pain

We recently demonstrated a contributing role of spinal cord infiltrating CD4+ T lymphocytes in the maintenance of mechanical hypersensitivity in a rodent model of neuropathic pain, spinal nerve L5 transection (L5Tx). It has been demonstrated that microglia play a role in the etiology of pain states. We hypothesized that infiltrating CD4+ T lymphocytes communicate with microglia via a CD40‐CD154 interaction. Here, we investigated the role of CD40 in the development of mechanical hypersensitivity post‐L5Tx. CD40 KO mice displayed significantly decreased mechanical sensitivity compared with WT mice starting from day 5 post‐L5Tx. Using bone marrow chimeric mice, we further identified a pro‐nociceptive role of CNS microglial CD40 rather than the peripheral leukocytic CD40. Flow cytometric analysis determined a significant increase of CD40+ microglia in the ipsilateral side of lumbar spinal cord post‐L5Tx. Further, spinal cord proinflammatory cytokine (IL‐1β, IL‐6, IL‐12, and TNF‐α) profiling demonstrated an induction of IL‐6 in both WT and CD40 KO mice post‐L5Tx prior to the increase of microglial CD40 expression, indicating a CD40‐independent induction of IL‐6 following L5Tx. These data establish a novel role of microglial CD40 in the maintenance of nerve injury‐induced behavioral hypersensitivity, a behavioral sign of neuropathic pain.

Propentofylline Targets TROY, a Novel Microglial Signaling Pathway

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain cancer, with a median survival of less than 2 years after diagnosis with current available therapies. The tumor microenvironment serves a critical role in tumor invasion and progression, with microglia as a critical player. Our laboratory has previously demonstrated that propentofylline, an atypical methylxanthine with central nervous system glial modulating and anti-inflammatory actions, significantly decreases tumor growth in a GBM rodent model by preferentially targeting microglia. In the present study, we used the CNS-1 rat glioma model to elucidate the mechanisms of propentofylline. Here we demonstrate that propentofylline targets TROY, a novel signaling molecule up-regulated in infiltrating microglia, and not macrophages, in response to CNS-1 cells. We identify Pyk2, Rac1 and pJNK as the downstream signaling molecules of TROY through western blot analysis and siRNA transfection. We demonstrate that inhibition of TROY expression in microglia by siRNA transfection significantly inhibits microglial migration towards CNS-1 cells similar to 10 µM propentofylline treatment. These results identify TROY as a novel molecule expressed in microglia, involved in their migration and targeted by propentofylline. Furthermore, these results describe a signaling molecule that is differentially expressed between microglia and macrophages in the tumor microenvironment.