Djane B. Duarte

Suppression of inflammatory and neuropathic pain by uncoupling CRMP-2 from the presynaptic Ca2+ channel complex

The use of N-type voltage-gated calcium channel (CaV2.2) blockers to treat pain is limited by many physiological side effects. Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function. A peptide of CRMP-2 fused to the HIV transactivator of transcription (TAT) protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, reduced meningeal blood flow, reduced nocifensive behavior induced by formalin injection or corneal capsaicin application and reversed neuropathic hypersensitivity produced by an antiretroviral drug. TAT-CBD3 was mildly anxiolytic without affecting memory retrieval, sensorimotor function or depression. At doses tenfold higher than that required to reduce hypersensitivity in vivo, TAT-CBD3 caused a transient episode of tail kinking and body contortion. By preventing CRMP-2–mediated enhancement of CaV2.2 function, TAT-CBD3 alleviated inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing chronic pain.

Models of Inflammation: Carrageenan‐ or Complete Freund's Adjuvant (CFA)–Induced Edema and Hypersensitivity in the Rat

Animal models of inflammation are used to assess the production of inflammatory mediators at sites of inflammation, the anti‐inflammatory properties of agents such as nonsteroidal anti‐inflammatory drugs (NSAIDs), and the efficacy of putative analgesic compounds in reversing cutaneous hypersensitivity. This unit details methods to elicit and measure carrageenan‐ and complete Freunds adjuvant (CFA)–induced cutaneous inflammation. Due to possible differences between the dorsal root sensory system and the trigeminal sensory system, injections of either the footpad or vibrissal pad are described. In this manner, cutaneous inflammation can be assessed in tissue innervated by the lumbar dorsal root ganglion neurons (footpad) and by the trigeminal ganglion neurons (vibrissal pad). Curr. Protoc. Pharmacol. 56:5.4.1‐5.4.7.

Models of Inflammation: Carrageenan Air Pouch

The subcutaneous air pouch is an in vivo model that can be used to study acute and chronic inflammation, the resolution of the inflammatory response, and the oxidative stress response. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this unit has been extensively used to identify potential anti‐inflammatory drugs. Curr. Protoc. Pharmacol. 56:5.6.1‐5.6.8.

Signaling pathways that mediate nerve growth factor-induced increase in expression and release of calcitonin gene-related peptide from sensory neurons.

Nerve growth factor (NGF) can augment transmitter release in sensory neurons by acutely sensitizing sensory neurons and by increasing the expression of calcitonin gene-related peptide (CGRP) over time. The current study examined the intracellular signaling pathways that mediate these two temporally distinct effects of NGF to augment CGRP release from sensory neurons. Growing sensory neurons in 30 or 100 ng/mL of NGF for 7 days increases CGRP content and this increase augments the amount of CGRP that is released by high extracellular potassium. Overexpressing a dominant negative Ras, Ras(17N) or treatment with a farnesyltransferase inhibitor attenuates the NGF-induced increase in CGRP content. Conversely, overexpressing a constitutively active Ras augments the NGF-induced increase in content of CGRP. Inhibiting mitogen activated protein kinase (MEK) activity also blocks the ability of NGF to increase CGRP expression. In contrast to the ability of chronic NGF to increase peptide content, acute exposure of sensory neurons to 100 ng/mL NGF augments capsaicin-evoked release of CGRP without affecting the content of CGRP. This sensitizing action of NGF is not affected by inhibiting Ras, MEK, or PI3 kinases. In contrast, the NGF-induced increase in capsaicin-evoked release of CGRP is blocked by the protein kinase C (PKC) inhibitor, BIM and the Src family kinases inhibitor, PP2. These data demonstrate that different signaling pathways mediate the alterations in expression of CGRP by chronic NGF and the acute actions of the neurotrophin to augment capsaicin-evoked release of CGRP in the absence of a change in the content of the peptide.

The Role of DNA Damage and Repair in Neurotoxicity Caused by Cancer Therapies

Abundant evidence in patients and in animal models demonstrates that cancer therapies have two major neurological side effects: cognitive dysfunction commonly termed “chemobrain” and chemotherapy-induced peripheral neuropathy (CIPN). Although a number of cancer therapies result in neurotoxicity, this chapter focuses on ionizing radiation and drugs that act through causing DNA damage. To date, the cellular mechanisms for cognitive dysfunction or CIPN have not been identified. Furthermore, there are no standard and effective treatments available to prevent or reverse therapy-induced neurotoxicity. One promising area that requires further exploration is the ability of DNA repair mechanisms to reverse the neurotoxic effects of a number of anticancer drugs. Studies in animal models show that enhancing the base excision repair pathway attenuates neuronal damage by chemotherapeutic agents, suggesting that manipulating DNA repair mechanisms may be a novel approach to diminish neurotoxicity during or after cancer therapy.

Reduced expression of SynGAP, a neuronal GTPase-activating protein, enhances capsaicin-induced peripheral sensitization

Synaptic GTPase-activating protein (SynGAP) is a neuronal-specific Ras/Rap-GAP that increases the hydrolysis rate of GTP to GDP, converting Ras/Rap from the active into the inactive form. The Ras protein family modulates a wide range of cellular pathways including those involved in sensitization of sensory neurons. Since GAPs regulate Ras activity, SynGAP might be an important regulator of peripheral sensitization and pain. Therefore, we evaluated excitability, stimulus-evoked release of the neuropeptide calcitonin gene-related peptide (CGRP), and nociception from wild-type (WT) mice and those with a heterozygous mutation of the SynGAP gene (SynGAP(+/-)). Our results demonstrate that SynGAP is expressed in primary afferent sensory neurons and that the capsaicin-stimulated CGRP release from spinal cord slices was two-fold higher from SynGAP(+/-) mice than that observed from WT mouse tissue, consistent with an increase in expression of the capsaicin receptor, transient receptor potential cation channel subfamily V member 1 (TRPV1), in SynGAP(+/-) dorsal root ganglia. However, there was no difference between the two genotypes in potassium-stimulated release of CGRP, the number of action potentials generated by a ramp of depolarizing current, or mechanical hypernociception elicited by intraplantar injection of capsaicin. In contrast, capsaicin-induced thermal hypernociception occurred at lower doses of capsaicin and had a longer duration in SynGAP(+/-) mice than WT mice. These results provide the first evidence that SynGAP is an important regulator of neuropeptide release from primary sensory neurons and can modulate capsaicin-induced hypernociception, demonstrating the importance of GAP regulation in signaling pathways that play a role in peripheral sensitization.