Vivianne L. Tawfik

Neuroimmune Activation and Neuroinflammation in Chronic Pain and Opioid Tolerance/Hyperalgesia:

One area that has emerged as a promising therapeutic target for the treatment and prevention of chronic pain and opioid tolerance/hyperalgesia is the modulation of the central nervous system (CNS) immunological response that ensues following injury or opioid administration. Broadly defined, central neuroimmune activation involves the activation of cells that interface with the peripheral nervous system and blood. Activation of these cells, as well as parenchymal microglia and astrocytes by injury, opioids, and other stressors, leads to subsequent production of cytokines, cellular adhesion molecules, chemokines, and the expression of surface antigens that enhance a CNS immune cascade. This response can lead to the production of numerous pain mediators that can sensitize and lower the threshold of neuronal firing: the pathologic correlate to central sensitization and chronic pain states. CNS innate immunity and Toll-like receptors, in particular, may be vital players in this orchestrated immune response and may hold the answers to what initiates this complex cascade. The challenge remains in the careful perturbation of injury/opioid-induced neuroimmune activation to down-regulate this process without inhibiting beneficial CNS autoimmunity that subserves neuronal protection following injury.

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

Deep Brain Stimulation Results in Local Glutamate and Adenosine Release: Investigation into the Role of Astrocytes

BACKGROUNDSeveral neurological disorders are treated with deep brain stimulation; however, the mechanism underlying its ability to abolish oscillatory phenomena associated with diseases as diverse as Parkinsons disease and epilepsy remain largely unknown. OBJECTIVETo investigate the role of specific neurotransmitters in deep brain stimulation and determine the role of non-neuronal cells in its mechanism of action. METHODSWe used the ferret thalamic slice preparation in vitro, which exhibits spontaneous spindle oscillations, to determine the effect of high-frequency stimulation on neurotransmitter release. We then performed experiments using an in vitro astrocyte culture to investigate the role of glial transmitter release in high-frequency stimulation-mediated abolishment of spindle oscillations. RESULTSIn this series of experiments, we demonstrated that glutamate and adenosine release in ferret slices was able to abolish spontaneous spindle oscillations. The glutamate release was still evoked in the presence of the Na+ channel blocker tetrodotoxin, but was eliminated with the vesicular H+-ATPase inhibitor bafilomycin and the calcium chelator 2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis acetoxymethyl ester. Furthermore, electrical stimulation of purified primary astrocytic cultures was able to evoke intracellular calcium transients and glutamate release, and bath application of 2-bis (2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid tetrakis acetoxymethyl ester inhibited glutamate release in this setting. CONCLUSIONVesicular astrocytic neurotransmitter release may be an important mechanism by which deep brain stimulation is able to achieve clinical benefits.

Propentofylline-Induced Astrocyte Modulation Leads to Alterations in Glial Glutamate Promoter Activation Following Spinal Nerve Transection

We have previously shown that the atypical methylxanthine, propentofylline, reduces mechanical allodynia after peripheral nerve transection in a rodent model of neuropathy. In the present study, we sought to determine whether propentofylline-induced glial modulation alters spinal glutamate transporters, glutamate transporter-1 (GLT-1) and glutamate-aspartate transporter (GLAST) in vivo, which may contribute to reduced behavioral hypersensitivity after nerve injury. In order to specifically examine the expression of the spinal glutamate transporters, a novel line of double transgenic GLT-1-enhanced green fluorescent protein (eGFP)/GLAST-Discosoma Red (DsRed) promoter mice was used. Adult mice received propentofylline (10 mg/kg) or saline via i.p. injection starting 1 h prior to L5-spinal nerve transection and then daily for 12 days. Mice receiving saline exhibited punctate expression of both eGFP (GLT-1 promoter activation) and DsRed (GLAST promoter activation) in the dorsal horn of the spinal cord, which was decreased ipsilateral to nerve injury on day 12. Propentofylline administration reinstated promoter activation on the injured side as evidenced by an equal number of eGFP (GLT-1) and DsRed (GLAST) puncta in both dorsal horns. As demonstrated in previous studies, propentofylline induced a concomitant reversal of L5 spinal nerve transection-induced expression of glial fibrillary acidic protein (GFAP). The ability of propentofylline to alter glial glutamate transporters highlights the importance of controlling aberrant glial activation in neuropathic pain and suggests one possible mechanism for the anti-allodynic action of this drug.

Induction of astrocyte differentiation by propentofylline increases glutamate transporter expression in vitro: Heterogeneity of the quiescent phenotype

Reactive astrocytes display decreased glutamate transporters, such as GLT‐1, and as a result synaptic glutamate clearance is impaired. In addition, these activated astrocytes are immunocompetent and release algesic mediators that can sensitize neurons in the spinal cord. Currently, we evaluated the effect of propentofylline (PPF), an experimental antiallodynic agent, on the phenotype and glutamate transporter expression of astrocytes. Primary astrocyte cultures, which represent an activated phenotype with a polygonal morphology and low GLT‐1 expression, were treated for 3 or 7 days with 10, 100, or 1,000 μM PPF or dibutyryl‐cAMP (db‐cAMP), a known inducer of GLT‐1 expression. PPF dose‐dependently induced astrocytes to display a mature phenotype, with elongated processes and a stellate shape, as well as increased GLT‐1 and GLAST immunoreactivity, similar to that seen with db‐cAMP. Real time RT‐PCR and Western blot analysis clearly demonstrated that PPF caused a potent dose‐dependent induction of GLT‐1 and GLAST mRNA and protein in these astrocytes. Importantly, the observed increase in glutamate transporters was found to have a functional effect, with significantly enhanced glutamate uptake in astrocytes treated with 100 or 1,000 μM PPF that was sensitive to dihydrokainate inhibition, suggesting it is GLT‐1 mediated. Finally, the effect of PPF on lipopolysaccaride‐induced chemokine release was investigated. Interestingly, PPF was able to dampen both MCP‐1 (CCL2) and MIP‐2 (CXCL2) release from astrocytes while db‐cAMP significantly enhanced this chemokine expression. These findings suggest that PPF is capable of differentiating astrocytes to a homeostatic, mature phenotype, competent for glutamate clearance and distinct from that induced by db‐cAMP.

Differential Spinal Cord Gene Expression in Rodent Models of Radicular and Neuropathic Pain

Background:Neuropathic pain and radicular low back pain both have a major impact on human health worldwide. Microarray gene analysis on central nervous system tissues holds great promise for discovering novel targets for persistent pain modulation. Methods:Rat models of lumbar radiculopathy (L5 nerve root ligation) and neuropathy (L5 spinal nerve transection) were used for these studies. The authors measured mechanical allodynia followed by analysis of global gene expression in the lumbar spinal cord at two time points (7 days and 14 days) after surgery using the Affymetrix RAE230A GeneChip® (Santa Clara, CA). The expression patterns of several genes of interest were subsequently confirmed using real-time reverse transcriptase polymerase chain reaction. Results:The authors observed similarly robust mechanical allodynia in both models. Second, they observed significant differences in lumbar spinal cord gene expression across chronic pain models. There was little overlap between genes altered in each injury model, suggesting that the site and type of injury produce distinct spinal mechanisms mediating the observed mechanical allodynia. The authors further confirmed a subset of the genes using reverse transcriptase polymerase chain reaction and identified several genes as either neuropathy-associated genes or radiculopathy-associated genes. Conclusions:These two models of persistent pain produce similar allodynic outcomes but produce differential gene expression. These results suggest that diverging mechanisms lead to a common behavioral outcome in these pain models. Furthermore, these distinct pathophysiologic mechanisms in neuropathic versus radicular pain may implicate unique drug therapies for these types of chronic pain syndromes.

Loss of [mu] opioid receptor signaling in nociceptors, but not microglia, abrogates morphine tolerance without disrupting analgesia

Opioid pain medications have detrimental side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). Tolerance and OIH counteract opioid analgesia and drive dose escalation. The cell types and receptors on which opioids act to initiate these maladaptive processes remain disputed, which has prevented the development of therapies to maximize and sustain opioid analgesic efficacy. We found that μ opioid receptors (MORs) expressed by primary afferent nociceptors initiate tolerance and OIH development. RNA sequencing and histological analysis revealed that MORs are expressed by nociceptors, but not by spinal microglia. Deletion of MORs specifically in nociceptors eliminated morphine tolerance, OIH and pronociceptive synaptic long-term potentiation without altering antinociception. Furthermore, we found that co-administration of methylnaltrexone bromide, a peripherally restricted MOR antagonist, was sufficient to abrogate morphine tolerance and OIH without diminishing antinociception in perioperative and chronic pain models. Collectively, our data support the idea that opioid agonists can be combined with peripheral MOR antagonists to limit analgesic tolerance and OIH.

The organizational and activational effects of sex hormones on tactile and thermal hypersensitivity following lumbar nerve root injury in male and female rats.

&NA; Considerable evidence exists for sex differences in human pain sensitivity. Women typically report a higher incidence of various painful conditions and report that the conditions are more painful when compared to men. In the present study, we sought to determine whether sex differences in pain sensitivity are observed using a lumbar radiculopathy model of low back pain in the rat and whether removal or alteration of gonadal hormones at specific timepoints can modulate these sex differences. Pubertal and adult male and female Sprague–Dawley rats were castrated 2 or 6 weeks prior to L5 nerve root injury to determine the activational hormonal effects. In a separate study, neonatal male and female Sprague–Dawley rats were either castrated or injected with testosterone, respectively, on postnatal day one to determine the organizational effects of gonadal hormones on L5 nerve root injury‐induced behavioral hypersensitivity. Our results demonstrate that there was a statistically significant sex difference in the magnitude of mechanical allodynia and thermal hyperalgesia following experimentally induced radiculopathy in the rat: females demonstrated decreased thresholds to tactile and thermal stimuli as compared to males. Furthermore, the enhanced female hypersensitivity was reversed in pubertal and adult animals ovariectomized 6 weeks, but not 2 weeks prior to L5 nerve root injury. Our results demonstrate that the activational effects of gonadal hormones mediate the enhanced female tactile and thermal hypersensitivity following L5 nerve root injury. These results suggest that manipulation of gonadal hormones may be a potential source for novel therapies for chronic pain in women.

Input- and cell-type-specific endocannabinoid-dependent LTD in the striatum.

Changes in basal ganglia plasticity at the corticostriatal and thalamostriatal levels are required for motor learning. Endocannabinoid-dependent long-term depression (eCB-LTD) is known to be a dominant form of synaptic plasticity expressed at these glutamatergic inputs; however, whether eCB-LTD can be induced at all inputs on all striatal neurons is still debatable. Using region-specific Cre mouse lines combined with optogenetic techniques, we directly investigated and distinguished between corticostriatal and thalamostriatal projections. We found that eCB-LTD was successfully induced at corticostriatal synapses, independent of postsynaptic striatal spiny projection neuron (SPN) subtype. Conversely, eCB-LTD was only nominally present at thalamostriatal synapses. This dichotomy was attributable to the minimal expression of cannabinoid type 1 (CB1) receptors on thalamostriatal terminals. Furthermore, coactivation of dopamine receptors on SPNs during LTD induction re-established SPN-subtype-dependent eCB-LTD. Altogether, our findings lay the groundwork for understanding corticostriatal and thalamostriatal synaptic plasticity and for striatal eCB-LTD in motor learning.

Safety issues concerning the medical use of cannabis and cannabinoids

Safety issues are a major barrier to the use of cannabis and cannabinoid medications for clinical purposes. Information on the safety of herbal cannabis may be derived from studies of recreational cannabis use, but cannabis exposure and effects may differ widely between medical and recreational cannabis users. Standardized, quality-controlled cannabinoid products are available in Canada, and safety profiles of approved medications are available through the Canadian formulary. In the present article, the evidence behind major safety issues related to cannabis use is summarized, with the aim of promoting informed dialogue between physicians and patients in whom cannabinoid therapy is being considered. Caution is advised in interpreting these data, because clinical experience with cannabinoid use is in the early stages. There is a need for long-term safety monitoring of patients using cannabinoids for a wide variety of conditions, to further guide therapeutic decisions and public policy.