Martina Kurejova

Hematopoietic colony–stimulating factors mediate tumor-nerve interactions and bone cancer pain

Pain is one of the most severe and debilitating symptoms associated with several forms of cancer. Various types of carcinomas and sarcomas metastasize to skeletal bones and cause spontaneous bone pain and hyperalgesia, which is accompanied by bone degradation and remodeling of peripheral nerves. Despite recent advances, the molecular mechanisms underlying the development and maintenance of cancer-evoked pain are not well understood. Several types of non-hematopoietic tumors secrete hematopoietic colony-stimulating factors that act on myeloid cells and tumor cells. Here we report that receptors and signaling mediators of granulocyte- and granulocyte-macrophage colony-stimulating factors (G-CSF and GM-CSF) are also functionally expressed on sensory nerves. GM-CSF sensitized nerves to mechanical stimuli in vitro and in vivo, potentiated CGRP release and caused sprouting of sensory nerve endings in the skin. Interruption of G-CSF and GM-CSF signaling in vivo led to reduced tumor growth and nerve remodeling, and abrogated bone cancer pain. The key significance of GM-CSF signaling in sensory neurons was revealed by an attenuation of tumor-evoked pain following a sensory nerve–specific knockdown of GM-CSF receptors. These results show that G-CSF and GM-CSF are important in tumor-nerve interactions and suggest that their receptors on primary afferent nerve fibers constitute potential therapeutic targets in cancer pain.

Presynaptically localized cyclic GMP-dependent protein kinase 1 is a key determinant of spinal synaptic potentiation and pain hypersensitivity.

Electrophysiological and behavioral experiments in mice reveal that a cGMP-dependent kinase amplifies neurotransmitter release from peripheral pain sensors, potentiates spinal synapses, and leads to exaggerated pain.

An improved behavioural assay demonstrates that ultrasound vocalizations constitute a reliable indicator of chronic cancer pain and neuropathic pain

BackgroundOn-going pain is one of the most debilitating symptoms associated with a variety of chronic pain disorders. An understanding of mechanisms underlying on-going pain, i.e. stimulus-independent pain has been hampered so far by a lack of behavioural parameters which enable studying it in experimental animals. Ultrasound vocalizations (USVs) have been proposed to correlate with pain evoked by an acute activation of nociceptors. However, literature on the utility of USVs as an indicator of chronic pain is very controversial. A majority of these inconsistencies arise from parameters confounding behavioural experiments, which include novelty, fear and stress due to restrain, amongst others.ResultsWe have developed an improved assay which overcomes these confounding factors and enables studying USVs in freely moving mice repetitively over several weeks. Using this improved assay, we report here that USVs increase significantly in mice with bone metastases-induced cancer pain or neuropathic pain for several weeks, in comparison to sham-treated mice. Importantly, analgesic drugs which are known to alleviate tumour pain or neuropathic pain in human patients significantly reduce USVs as well as mechanical allodynia in corresponding mouse models.ConclusionsWe show that studying USVs and mechanical allodynia in the same cohort of mice enables comparing the temporal progression of on-going pain (i.e. stimulus-independent pain) and stimulus-evoked pain in these clinically highly-relevant forms of chronic pain.

A Functional Role for VEGFR1 Expressed in Peripheral Sensory Neurons in Cancer Pain

Summary Cancer pain is a debilitating disorder and a primary determinant of the poor quality of life. Here, we report a non-vascular role for ligands of the Vascular Endothelial Growth Factor (VEGF) family in cancer pain. Tumor-derived VEGF-A, PLGF-2, and VEGF-B augment pain sensitivity through selective activation of VEGF receptor 1 (VEGFR1) expressed in sensory neurons in human cancer and mouse models. Sensory-neuron-specific genetic deletion/silencing or local or systemic blockade of VEGFR1 prevented tumor-induced nerve remodeling and attenuated cancer pain in diverse mouse models in vivo. These findings identify a therapeutic potential for VEGFR1-modifying drugs in cancer pain and suggest a palliative effect for VEGF/VEGFR1-targeting anti-angiogenic tumor therapies.

Pain in experimental autoimmune encephalitis: a comparative study between different mouse models

BackgroundPain can be one of the most severe symptoms associated with multiple sclerosis (MS) and develops with varying levels and time courses. MS-related pain is difficult to treat, since very little is known about the mechanisms underlying its development. Animal models of experimental autoimmune encephalomyelitis (EAE) mimic many aspects of MS and are well-suited to study underlying pathophysiological mechanisms. Yet, to date very little is known about the sensory abnormalities in different EAE models. We therefore aimed to thoroughly characterize pain behavior of the hindpaw in SJL and C57BL/6 mice immunized with PLP139-151 peptide or MOG35-55 peptide respectively. Moreover, we studied the activity of pain-related molecules and plasticity-related genes in the spinal cord and investigated functional changes in the peripheral nerves using electrophysiology.MethodsWe analyzed thermal and mechanical sensitivity of the hindpaw in both EAE models during the whole disease course. Qualitative and quantitative immunohistochemical analysis of pain-related molecules and plasticity-related genes was performed on spinal cord sections at different timepoints during the disease course. Moreover, we investigated functional changes in the peripheral nerves using electrophysiology.ResultsMice in both EAE models developed thermal hyperalgesia during the chronic phase of the disease. However, whereas SJL mice developed marked mechanical allodynia over the chronic phase of the disease, C57BL/6 mice developed only minor mechanical allodynia over the onset and peak phase of the disease. Interestingly, the magnitude of glial changes in the spinal cord was stronger in SJL mice than in C57BL/6 mice and their time course matched the temporal profile of mechanical hypersensitivity.ConclusionsDiverse EAE models bearing genetic, clinical and histopathological heterogeneity, show different profiles of sensory and pathological changes and thereby enable studying the mechanistic basis and the diversity of changes in pain perception that are associated with distinct types of MS.

Gαq/11 signaling tonically modulates nociceptor function and contributes to activity-dependent sensitization

TOC summary The functional role of Gq/11 G proteins in nociceptors not only spans pathological pain, but, surprisingly, also includes tonic modulation of nociception. ABSTRACT Peripheral injury or inflammation leads to a release of mediators capable of binding to a variety of ion channels and receptors. Among these are the 7‐transmembrane receptors (G protein‐coupled receptors) coupling to Gs, Gi/o, G12/13, or Gq/11 G proteins. Each of the G protein‐coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of individual signaling pathways in vivo has not yet been worked out. The Gq/G11 signaling branch is of particular interest because it leads to the activation of phospholipase C‐β, protein kinase C, the release of calcium from intracellular stores, and it modulates extracellular regulated kinases. To investigate the contribution of the entire Gq/11‐signaling pathway in nociceptors towards regulation of pain, we generated double‐deficient mice lacking Gq/11 selectively in nociceptors using a conditional gene‐targeting approach. We observed that nociceptor‐specific loss of Gq and G11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. Surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in Gq/11 mutant mice, suggesting a novel function for Gq/11 in tonic modulation of acute nociception. Patch‐clamp recordings revealed changes in voltage‐dependent tetrodotoxin‐resistant and tetrodotoxin‐sensitive sodium channels in nociceptors upon a loss of Gq/11, whereas potassium currents remained unchanged. Our results indicate that the functional role of the Gq/G11 branch of G‐protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain.

Conditional gene deletion reveals functional redundancy of GABAB receptors in peripheral nociceptors in vivo

Backgroundγ-aminobutyric acid (GABA) is an important inhibitory neurotransmitter which mainly mediates its effects on neurons via ionotropic (GABAA) and metabotropic (GABAB) receptors. GABAB receptors are widely expressed in the central and the peripheral nervous system. Although there is evidence for a key function of GABAB receptors in the modulation of pain, the relative contribution of peripherally- versus centrally-expressed GABAB receptors is unclear.ResultsIn order to elucidate the functional relevance of GABAB receptors expressed in peripheral nociceptive neurons in pain modulation we generated and analyzed conditional mouse mutants lacking functional GABAB(1) subunit specifically in nociceptors, preserving expression in the spinal cord and brain (SNS-GABAB(1)-/- mice). Lack of the GABAB(1) subunit precludes the assembly of functional GABAB receptor. We analyzed SNS-GABAB(1)-/- mice and their control littermates in several models of acute and neuropathic pain. Electrophysiological studies on peripheral afferents revealed higher firing frequencies in SNS-GABAB(1)-/- mice compared to corresponding control littermates. However no differences were seen in basal nociceptive sensitivity between these groups. The development of neuropathic and chronic inflammatory pain was similar across the two genotypes. The duration of nocifensive responses evoked by intraplantar formalin injection was prolonged in the SNS-GABAB(1)-/- animals as compared to their control littermates. Pharmacological experiments revealed that systemic baclofen-induced inhibition of formalin-induced nociceptive behaviors was not dependent upon GABAB(1) expression in nociceptors.ConclusionThis study addressed contribution of GABAB receptors expressed on primary afferent nociceptive fibers to the modulation of pain. We observed that neither the development of acute and chronic pain nor the analgesic effects of a systematically-delivered GABAB agonist was significantly changed upon a specific deletion of GABAB receptors from peripheral nociceptive neurons in vivo. This lets us conclude that GABAB receptors in the peripheral nervous system play a less important role than those in the central nervous system in the regulation of pain.

A novel biological role for the phospholipid lysophosphatidylinositol in nociceptive sensitization via activation of diverse G-protein signalling pathways in sensory nerves in vivo

Summary Lysophosphatidylinositol (LPI) is a novel regulator of peripheral sensory neuron function and pathological pain. The roles of GPCR and non‐GPCR components to the biological function of LPI are delineated. Abstract The rich diversity of lipids and the specific signalling pathways they recruit provides tremendous scope for modulation of biological functions. Lysophosphatidylinositol (LPI) is emerging as a key modulator of cell proliferation, migration, and function, and holds important pathophysiological implications due to its high levels in diseased tissues, such as in cancer. Here we report a novel role for LPI in sensitization of peripheral sensory neurons, which was evident as exaggerated sensitivity to painful and innocuous pressure. Histopathological analyses indicated lack of involvement of myelin pathology and immune cell recruitment by LPI. Using pharmacological and conditional genetic tools in mice, we delineated receptor‐mediated from non‐receptor‐mediated effects of LPI and we observed that GPR55, which functions as an LPI receptor when heterologously expressed in mammalian cells, only partially mediates LPI‐induced actions in the context of pain sensitization in vivo; we demonstrate that, in vivo, LPI functions by activating G&agr;13 as well as G&agr;q/11 arms of G‐protein signalling in sensory neurons. This study thus reports a novel pathophysiological function for LPI and elucidates underlying molecular mechanisms.

311 HEMATOPOIETIC COLONY STIMULATING FACTORS MEDIATE TUMOR-NERVE INTERACTIONS AND BONE CANCER PAIN

Pain is one of the most severe and debilitating symptoms associated with severalforms of cancer. Various types of carcinomas and sarcomas metastasize to skeletal bones and cause spontaneous bone pain and hyperalgesia, which is accompanied by bone degradation and remodeling of peripheral nerves. Despite recent advances, the molecular mechanisms underlying the development and maintenance of cancer-evoked pain are not well understood. Several types of non-hematopoietic tumors secrete hematopoietic colony stimulating factors which act on myeloid cells5 and tumor cells. Here we report that receptors and signaling mediators of granulocyteand granulocyte-macrophage colony stimulating factors (G-/GM-CSF) are also functionally expressed on primary afferent nerve fibers. GM-CSF sensitized nerve fibers to mechanical stimuli in vitro and in vivo, potentiated CGRP release and caused sprouting of sensory nerve endings in the skin. Interruption of G-/GM-CSF signaling in vivo led to reduced tumor growth and nerve remodeling, and abrogated bone cancer pain. The key significance of GM-CSF signaling in sensory neurons was revealed by an attenuation of tumor-evoked pain following a sensory nervespecific knockdown of GM-CSF receptors. These results show that G-/GM-CSF play an important role in tumor-nerve interactions and suggest that G-/GM-CSF receptors on primary afferent nerve fibers constitute important, novel therapeutic targets in cancer pain.