Part II

During nerve injury, pain is associated with release of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), and interleukin-6 (IL-6), which are essential for establishing nociceptive processing (Marchand et al., 2005, Fecho et al., 2007). TNF-α is a neuropathic pain-related cytokine, which has been found to have a lead role in activating a local cascade of other pro-inflammatory cytokines, such as IL-1β and IL-6, in a model of neuropathic pain following nerve injury (Shamash et al., 2002). However, there is as yet no common consensus about the roles of neuropathic pain - possible mechanisms can be categorized into peripheral sensitization and central sensitization of the nervous system in response to the nociceptive stimuli (Leung and Cahill, 2010). In addition, IL-6 has been associated with the development of neuropathic pain in various animal models, and high level of IL-1β has been found to be associated with CCI-induced microglial activation. Thus, the attenuation of pro-inflammatory cytokines results in alleviation of neuropathic pain. In the list of the pro-inflammatory cytokines, IL-1 appears to be a key mediator of the neuroinflammation. In fact, IL-1 has been reported to mediate many neurological effects in the spinal cord (Detloff et al., 2008) and brain (Rothwell, 1999). A relatively high level of IL-1β also has been found to be associated with TBI-induced neuron loss (Toulmond and Rothwell, 1995, Tehranian et al., 2002, Lu et al., 2005). Thus, an efficient method

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that could ultimately confer a decline in IL-1β and the traumatic inflammatory response is likely to be an attractive strategy for TBI treatment (Lynch et al., 2005, Chen et al., 2011).

1.4.1 Endogenic Opioid and neuropathic pain

Opioids are quite effective in fighting acute and chronic pain. In addition, opioids help regulate the immune system (Finley et al., 2008) and have neuroprotective properties (Berrios et al., 2008). Nevertheless, clinical exogenous opioid administration is associated with several side effects in addition to tolerance development because of their central mechanisms of action, thus limiting their use (Ugolini et al., 2007). To overcome these limitations, endogenous opioid-mediated antinociception has been extensively studied, and its physiological and clinical relevance have been established (Stein et al., 2003).

In both early inflammation and chronic neuropathic models, hyperalgesia can be partially counteracted by a local antinociceptive system involving opioid-containing leukocytes (Stein et al., 1990, Stein et al., 2003, Binder et al., 2004, Labuz et al., 2009, Busch-Dienstfertig and Stein, 2010). Under inflammatory conditions, leukocytes secrete opioid peptides that bind to opioid receptors on peripheral sensory neurons and mediate antinociception (Cabot et al., 2001, Brack et al., 2004b, Mousa et al., 2004, Rittner et al., 2008, Labuz et al., 2010). In humans, opioid peptides locally released by leukocytes can decrease pain intensity as well as the consumption of pain medication under post-surgical stress conditions (Stein et al., 2003). Because the majority of the

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opioid-containing leukocytes during early inflammation are polymorphonuclear (PMN) cells (Brack et al., 2004b, Rittner et al., 2006b), increasing the PMN cell within the inflammatory nerve is benefited for antinociception.

1.4.2 Granulocyte-colony stimulating factor (G-CSF)

As inflammation, treatment with granulocyte-colony stimulating factor (G-CSF) causes hematopoietic stem cell egression from bone marrow niches and mobilization to the peripheral blood (Hoggatt and Pelus, 2011). The G-CSF receptor (G-CSFR) is a transmembrane protein expressed on cells of the neutrophil lineage, including progenitor and differentiating myeloid cells in the bone marrow and mature neutrophils in the peripheral blood (Roberts, 2005). G-CSF then initiates precursor cell proliferation and differentiation into mature PMN cells (Touw and van de Geijn, 2007).

1.4.3 Nogo-A

Nogo-A, a myelin-rich membrane protein of the adult central nervous system (CNS), is known to act through specific binding to the Nogo receptor (NgR) (Fournier et al., 2001). Three isoforms of the Nogo protein (Nogo-A, Nogo-B, and Nogo-C) and of the corresponding NgRs have been identified (Venkatesh et al., 2005). The C-terminal sequences of all Nogo proteins bear a striking homology to several members of the reticulon or neuroendocrine-specific proteins, suggesting that Nogo-A is a

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member of the endoplasmic reticulum-anchored proteins. A growing body of studies has demonstrated that expression of Nogo-A is not restricted to neurons and oligodendrocytes in the CNS but occurs throughout the adult brain and the spinal cord (Huber et al., 2002, Hunt et al., 2003). It is a potent inhibitor of neurite outgrowth, and it is known to negatively regulate regeneration in the adult CNS (Chen et al., 2000, GrandPre et al., 2000). Treatment with anti-Nogo-A antibodies or an NgR antagonist can significantly promote axonal regeneration, neuroanatomical plasticity, and functional recovery (GrandPre et al., 2002, Seymour et al., 2005, Papadopoulos et al., 2006). Furthermore, recent studies have also demonstrated that the expression of Nogo-A and NgRs is stimulated by the activated microglia/macrophages (Fry et al., 2007). This converging evidence points to an important role for Nogo-A in mediating the inflammatory responses caused by various neurological conditions including TBI (David et al., 2008).

Endogenous CRF (Cabot et al., 1997, Cabot, 2001) and chemokines (ex. CXCL2/3) (Rittner et al., 2006a) expressed in inflamed tissue are prominent agents that trigger opioid peptides release from leukocytes, thereby inhibiting pain. Thus, G-CSF is an important factor for inducing the generation of new PMN cells, suggesting a potential beneficial role for treating inflammatory and chronic pain. Therefore, we proposed to administrate G-CSF to an animal model with neuropathic pain and evaluate whether G-CSF-induced activation of PMN cells can reverse expression of pro-inflammatory cytokines and chronic pain by releasing peripheral endogenous opioids. In addition, as the hippocampus was

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found to exhibit rather severe neuronal loss after brain injury (Lu et al., 2005, Lu et al., 2008), we sought to investigate cytokine-associated Nogo-A expression in CNS after TBI by treating with indomethacin.

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