以大鼠周邊及中樞神經損傷模式探討調節細胞激素對於神經系統的保護機制

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(1)國立臺灣師範大學生命科學系博士論文. 以大鼠周邊及中樞神經損傷模式探討調節細胞激素對於神經系統的保護機制 Elucidate the Neural Protective Mechanism of Regulation of Cytokines on the Peripheral Nerve Injury and Central Neuronal Damage Models in Rat. 研究生：趙伯寬 Po-Kuan Chao 指導教授：呂國棟博士 Kwok-Tung Lu, Ph. D. 共同指導：羅榮昇博士 Long-Sun Ro, Ph. D. 共同指導：楊奕玲博士 Yi-Ling Yang, Ph. D. 中華民國 101 年 7 月.

(2) PK Chao. Contents Abstract .................................................................................................... IV Abstract (Chinese) .................................................................................. VII Abbreviations ............................................................................................. X 1. Introduction .............................................................................................1 1.1 Neuropathic pain ...............................................................................1 1.2 Traumatic brain injury ......................................................................3 1.3 Part I ..................................................................................................4 1.4 Part II ................................................................................................8 2. Methods.................................................................................................13 2.1 Experimental animals .....................................................................13 2.2 Behavioral tests ...............................................................................16 2.3 Blood cell counting and flow cytometry ........................................17 2.4 IL-6 mRNA assay ...........................................................................18 2.5 Western blotting assay ....................................................................20 2.6 Enzyme-linked immunosorbent assay (ELISA) .............................21 2.7 Immunohistochemistry ...................................................................21 2.8 Statistical analysis ...........................................................................23 3. Results ...................................................................................................24 3.1 Part I ................................................................................................24 3.2 Part II ..............................................................................................28 4. Discussion .............................................................................................36 4.1 Part I ................................................................................................36 4.2 Part II ..............................................................................................45 5. Conclusions ...........................................................................................56. I.

(3) PK Chao. 6. References .............................................................................................58 7. Figures & Legends ................................................................................71 Figure 1 After unilateral CCI of the sciatic nerve, animals developed mechanical allodynia in both ipsi- and contra-lateral limbs..........71 Figure 2 The CCI operation mobilized immune cells into the injured nerves .............................................................................................73 Figure 3 c-Fos protein did not increase in the ipsilateral DRG after peripheral nerve injury ...................................................................74 Figure 4 c-Fos protein expressed in bilateral spinal DH of CCI rat .........75 Figure 5 CCI induces OX-42 positive immunoreactivity in bilateral spinal DH at 72 h after CCI…………………………………………….76 Figure 6 The number of OX-42 immunoreactive cells in the bilateral spinal DH correlated with mechanical paw withdrawal response of CCI rat at 72 h..………………………………………..……….. 78 Figure 7 The number of OX-42 immunoreactive cells in the contralateral spinal DH correlated with ipsilateral side of CCI rat at 72 h…... 79 Figure 8 G-CSF alleviates long-term thermal hyperalgesia and mechanical allodynia in rats with CCI ..........................................80 Figure 9 G-CSF demonstrates a therapeutic time window (0–48 h) after nerve injury………………………………………………………82 Figure 10 Effects of G-CSF on mobilized bone marrow cells in the peripheral blood. ............................................................................83 Figure 11 Effects of G-CSF on mobilized immune cells in the tissue fluid around the injured nerve ................................................................84 Figure 12 G-CSF treatment increases the number of migrated β-endorphin-containing PMN cells in injured nerves ...................86 Figure 13 μ-opioid receptor expressed in the sciatic nerve of both sham-operated and CCI-operated rat .............................................88. II.

(4) PK Chao. Figure 14 NLXM reverses the G-CSF anti-nociceptive effect .................89 Figure 15 G-CSF reduces the levels of IL-6 mRNA and TNF-α protein in the DRG after CCI .........................................................................91 Figure 16 Expression of IL-1β protein is up-regulated in ipsilateral spinal DH of vehicle- treated CCI rat ......................................................93 Figure 17 G-CSF reduces immunoreactivity of OX-42 in the spinal cord 72 h after CCI ................................................................................94 8. Figures and Legends of References ......................................................96 Reference figure 1 Modulation of neuropathic pain by immune cells. ....96. III.

(5) PK Chao. Abstract Recent studies have shown that modulation of cytokine expression is helpful to reduce nerve inflammation and to improve dysfunction of nerve system. In this study, we use rat models to investigate the mediators for alterations of cytokines expression following peripheral nerve injury and central neural damage. First, in order to clarify the correlation between histopathological assessment and behavior outcome, c-Fos protein expression and microglia activation were quantified in both of the ipsi- and contra-lateral spinal dorsal horn (DH) and/or Lumbar 5 (L5) dorsal root ganglia (DRG) in sham-operation and chronic constriction injury (CCI) rats. We found that c-Fos protein expression did not increase in the ipsilateral DRG of CCI rat. In addition, CCI evoked increase of c-Fos protein expression was only in the ipsilateral spinal DH after 72h. In contrast, however, microglia activation was notably induced at bilateral spinal DH and correlated with mechanical allodynia. Thus, neurons are not the only cells playing a role in neuropathic pain, we evidence that microglia are involved as well and there is a cross effect between neuron and microglia in the central nervous system (CNS) which associated to nociceptive behaviors. The current studies show that granulocyte colony-stimulating factor (G-CSF) is an important mediator of nerve system. Secondary, this study shows the effectiveness of exogenous treatment for alleviating thermal hyperalgesia and mechanical allodynia in rats with CCI, during post-operative days 1–25, compared to that of vehicle treatment. G-CSF. IV.

(6) PK Chao. also increases the recruitment of opioid-containing PMN cells into the injured nerve. After CCI, single administration of G-CSF, relieved thermal hyperalgesia, indicated that its effect on neuropathic pain had a therapeutic window of 0–48 h after nerve injury. CCI led to increase in the levels of interleukin-6 (IL-6) mRNA and tumor necrosis factor-alpha (TNF-α) protein in the DRG and interleukin-1beta (IL-1β) protein in the spinal cord. These high levels of IL-6 mRNA, TNF-α and IL-1β were, respectively, suppressed by a single administration of G-CSF after CCI, respectively. Moreover, the μ-opioid receptor was observed in injured nerve and opioid receptor antagonist naloxone methiodide (NLXM) reversed G-CSF-induced antinociception after CCI, suggesting that G-CSF alleviates hyperalgesia via opioid/opioid receptor interactions. Furthermore, G-CSF administered after CCI suppressed the CCI-induced up-regulation of microglial activation in the ipsilateral spinal DH, which is essential for sensing neuropathic pain. These results suggest that an early single systemic injection of G-CSF alleviates neuropathic pain via activation of PMN cell-derived endogenous opioid secretion to activate opioid receptors in the injured nerve, down-regulate IL-6, IL-1β and TNF- pro-inflammatory cytokines, and attenuate microglial activation in the spinal DH. In the neuronal damaged models, indomethacin was ever used to deliver a traumatic management to rats. Based on Western blot analyses, the expression of Nogo-A was found to be significantly up-regulated in the hippocampus beginning eight hours after traumatic brain injury (TBI). In addition, TBI caused an apparent elevation in IL-1β levels in the tested V.

(7) PK Chao. animals. All of the TBI-associated molecular and cellular consequences could be effectively reversed by treating the animals with the anti-inflammatory. drug. indomethacin.. More. importantly,. the. TBI-associated stimulation in the levels of both Nogo-A and IL-1β could be effectively inhibited by a specific Nogo-A antisense oligonucleotide. Our findings suggest that the suppression of Nogo-A expression appears to be an early response conferred by indomethacin, which then leads to decreases in the levels of IL-1β and TBI-induced neuron damage. In conclusion, because systemic administration of G-CSF or indomethacin had positive effects in both models of peripheral nerve injury and central nervous system injury in rats, respectively, it is highly possible that treating with G-CSF or indomethacin to modulate pro-inflammatory cytokines of CNS is the new method to manage central sensitization.. Keywords: granulocyte colony-stimulating factor, tumor necrosis factor, microglia, nerve injury, neuropathic pain, opioid, cytokine.. VI.

(8) PK Chao. Abstract (Chinese) 近年來的研究顯示，調節細胞激素有助於減少神經損傷後的神經發炎並且改善神經系統功能。在本研究當中，我們分別使用不同的大鼠模式來研究如何透過特定調節物質來改變周邊神經損傷及中樞神經傷害後細胞激素的表現。首先，為了釐清組織病理評估結果與行為結果之間的關聯性，我們針對假手術對照組及長期壓迫神經損傷手術組大鼠的同側及對側脊髓神經背角及第五段腰椎的背根神經結，分別測量其c-Fos蛋白質表現以及小膠細胞活化作用。我們發現c-Fos蛋白質表現在長期壓迫神經損傷手術組大鼠的同側背根神經結並不會增加。此外，手術後72 小時長期壓迫神經損傷引發c-Fos蛋白質表現增加只會出現在同側脊髓神經背角中。然而相對的，小膠細胞活化現象在脊髓神經背角兩側都非常顯著，同時也與機械性刺激引發痛覺過敏的行為結果一致。所以我們證實不僅有神經細胞、小膠細胞也在神經病變痛當中扮演角色，在中樞神經系統中，小膠細胞透過與神經細胞之間的交互作用對疼痛行為產生影響。先前的研究中發現在神經系統中顆粒細胞刺激增生因子是一種重要的調節物質。所以本研究展示了與對照組相較的結果，在手術後. VII.

(9) PK Chao. 的1到25天之間，投予顆粒細胞刺激增生因子有助於減輕長期壓迫神經損傷引發的熱痛覺過敏及機械性刺激過敏的現象。長期壓迫神經損傷手術手術後投予顆粒細胞刺激增生因子也減少動物熱痛覺過敏。推測若神經損傷後48小時內單次投與顆粒細胞刺激增生因子仍對於神經病變痛的治療具有效果。顆粒細胞刺激增生因子也驅動含鴉片類物質的聚多核細胞聚集進入受損的神經之中。長期壓迫神經損傷不僅導致背根神經結中的第六型細胞激素的mRNA及腫瘤壞死因子α亞型蛋白表現量增加，也使得脊髓中第一型細胞激素的蛋白表現量上升。這些細胞激素的產生都會被顆粒細胞刺激增生因子所抑制。我們更進一步發現長期壓迫神經損傷後，μ型鴉片受體出現在損傷的神經當中而且鴉片受體拮抗劑─naloxone methiodide減低了顆粒細胞刺激增生因子的抗痛效果，於是我們推論顆粒細胞刺激增生因子減輕熱痛覺過敏是透過鴉片類物質及其受體之間交互作用的結果。更進一步來看，投與顆粒細胞刺激增生因子可以抑制脊髓神經背角處因長期壓迫神經損傷所引發小膠細胞大量活化。這些結果推測在神經損傷初期單次全身性投予顆粒細胞刺激增生因子減輕神經病變痛的效果是透過活化聚多核細胞驅動的內生性鴉片類物質分泌，進而活化損傷神經當中的鴉片類物質受體，同時減少促發炎細胞激素，並減輕脊髓神經背角當. VIII.

(10) PK Chao. 中的小膠細胞活化現象。而在大鼠神經損傷模式中，indomethacin曾被用來進行創傷的治療。基於西方墨漬法的分析結果, 我們發現Nogo-A蛋白表現在頭部創傷後八小時顯著增加。此外，頭部創傷引發受測動物的第一型細胞激素含量顯著提升，一般而言所有的頭部創傷相關的分子及細胞的後續反應都會被抗發炎藥物indomethacin所影響而顯著減少。更重要的是，頭部創傷刺激作用相關的Nogo-A及第一型細胞激素的含量明顯被具有專一性的反義寡核苷酸所抑制。我們的發現，出現Nogo-A表現受到抑制的初期反應是由indomethacin所賦予的，也減少第一型細胞激素的表現程度並且減輕頭部誘發的神經損傷。總結以上，由於全身性投予顆粒細胞刺激增生因子或是 indomethacin分別對於大鼠的周邊以及中樞神經損傷具有正面的影響，以顆粒細胞刺激增生因子或者是indomethacin來調節中樞神經系統中的促發炎細胞激素，很可能有成為治療中樞神經過敏化現象的嶄新療法。. 關鍵字：顆粒細胞刺激增生因子、腫瘤壞死因子、小膠細胞、神經損傷、神經病變痛、鴉片類物質、細胞激素。. IX.

(11) PK Chao. Abbreviations. BBB. blood brain barrier. BSA. bovine serum albumin. CCI. chronic constriction injury. CNS. central nervous system. CRF. corticotropin-releasing factor. DEPC. diethylpyrocarbonate. DH. dorsal horn. DRG. dorsal root ganglia. EDTA. ethylenediaminetetraacetic acid. ELISA. enzyme-linked immunosorbent assay. ERK. extracellular signal-regulated kinase. FACS. fluorescence-activated cell sorting. FITC. fluorescein isothiocyanate. GAPDH. glyceraldehyde 3-phosphate dehydrogenase. G-CSF. granulocyte colony-stimulating factor. G-CSFR. granulocyte colony-stimulating factor receptor. HRP. horseradish peroxide. ICAM-1. intercellular adhesion molecule-1. i.p.. intraperitoneal. i.v.. intravenous. IL-1β. interleukin-1β. IL-6. interleukin-6 X.

(12) PK Chao. IL-10. interleukin-10. L4. lumbar 4. L5. lumbar 5. L6. lumbar 6. mAbs. monoclonal antibodies. MAPK. mitogen-activated protein kinase. NgR. Nogo receptor. NLXM. naloxone methiodide. Nogo-A,. Nogo protein A. Nogo-B,. Nogo protein B. Nogo-C. Nogo protein C. PBS. phosphate-buffered saline. PCR. polymerase chain reaction. PE. phycoerythrin. PGE2. prostaglandin E2. PMN. polymorphonuclear cell. PMSP. phenylmethylsulfonyl fluoride. PNS. Peripheral nervous system. PVDF. polyvinylidene fluoride. ROS. reactive oxygen species. s.c.. subcutaneous. SDS-PAGE. sodium dodecyl sulfate polyacrylamide gel electrophoresis. TBI. traumatic brain injury. XI.

(13) PK Chao. TNF-α. tumor necrosis factor-alpha. TRPV. transient receptor potential vanilloid. WHO. world health organization. XII.

(14) 1. Introduction. In the life cycle of human, people more or less suffer from some injury following an accident. Sometimes, tissue in the damaged site could get a complete recover after management. However, when people get a certain degree trauma, a part of the injured tissues could heal but a part of those could not be recovered and may lose the normal functions. Especially, in the nerve tissue, a severe trauma always due to a proportion of the injured people has lifelong sequelae. For example, in the peripheral nervous system (PNS), trauma triggers excitation of nociceptors and transmission of pain signals on the following inflammation. In some conditions, peripheral nerve injury inducing pain can be a direct or an indirect consequence of a neurological disorder, which possibly lead to neuropathic pain, such as multiple sclerosis or injury from an accident.. 1.1 Neuropathic pain Pain managements and treatments can be simple or complex, which depend on their causes. Since there are no established procedures and as a result, neuropathic pain often goes untreated or gets unsuitable managements. However, a current research found that the annual cost of pain was greater than the annual costs of heart disease ($309 billion), cancer ($243 billion), and diabetes ($188 billion) in the USA (Gaskin and Richard, 2012). This shows that there are many people suffer from neuropathic pain and a huge market of pain management was hided under 1.

(15) PK Chao. the table. Neuropathic or nerve pain is a type of pain that results either from damage to the nerves that normally sense pain or from injury to a part of the nervous system that transmits pain signals, such as the spinal cord or the brain. Neuronal mechanisms responsible for neuropathic pain include ectopic impulse generation, degeneration of sensory fibers, their sprouting to areas they normally do not innervate, sympathetic nerve sprouting, disinhibition, enhanced activity of descending facilitatory or impaired activity of descending inhibitory transmission (Leonardo et al., 2009). Because neuropathic pain may persist long after initial tissue damage has healed: in such cases, it becomes a speciﬁc health-care problem and a difficult recognized disease. There are a variety of methods to treat chronic pain, which are dependent on the types of pain experienced. When patients do not have a satisfactory response to the firstline medications alone or in combination, opioid agonists can be used as second-line treatment alone or in combination with the first-line medications (Dworkin et al., 2007). However, opioids have produced side effects more frequently than first-line medications, some of these side effects can persist throughout long-term treatment (Dworkin et al., 2007). In addition, the long-term safety of opioid treatment has not been systematically studied (Dworkin et al., 2007). A recent market research report indicates that more than 1.5 billion people. worldwide. suffer. from. chronic. neuropathic. pain,. that. approximately 3 to 4.5% of the global population suffers from 2.

(16) PK Chao. neuropathic pain. (Global Industry Analysts, Inc. Report, January 10, 2011). In addition, pain in about 70% of older patients is neuropathic pain and global population suffers from neuropathic pain with incidence rate increasing in complementary to age. These showed that, in the future, we would be likely to face the threat of neuropathic pain. However, in current studies, neuropathic pain is invalidating and less researched than the other neurodegeneration disease.. 1.2 Traumatic brain injury Traumatic brain injury (TBI) is one of the most prevalent traumas of central nervous system (CNS) in worldwide and causes neurological dysfunction and death through both primary and secondary cellular mechanisms. In most case, die as a result of a TBI, which was occurring at the primary stage of injury. Even person survive from the secondary response of TBI, those people become permanently disabled and have to endure lifelong debilitating loss of function. An additional case of those sustaining a TBI will exist in a persistent vegetative state. Thus, the treatment of TBI can result in enormous medical resource waste and huge social expenses. Both of the peripheral nerve injury and central nervous system injury induced inflammatory responses were associated with not only neuronal cell but also glia cell and immune cell (Watkins and Maier, 2002). The inflammatory response triggered by peripheral nerve injury was demonstrated to be associated to neuropathic pain and central sensitization (Costigan et al., 2009) and is characterized by glial 3.

(17) PK Chao. activation, leukocyte infiltration, and up-regulation of cytokine secretion (DeLeo et al., 2004). Similarly, the inflammatory response triggered by TBI was demonstrated to be closely related to neuron death and functional deficits (Cernak, 2005) and is characterized by glial activation, leukocyte recruitment, and up-regulation of cytokine secretion (Donnelly and Popovich, 2008). In our present study, we evidenced the effects of novel treatments, which improved the inflammation and neuropathic pain of peripheral nerve injury and central nervous system injury by regulating the pro-inflammatory cytokines.. 1.3 Part I. 1.3.1 Background The pathophysiology of neuroinflammation appears to be very complex and remains poorly understood. According to previous studies, peripheral nerve injury-induced long-term neuroinflammation could result in central sensitization and chronic nociceptive behaviors (Sotgiu et al., 1998). Instead, neuropathic pain may result from early altered in biochemical properties (George et al., 1999). Recent advances in the understanding of the mechanisms of ongoing pathological pain have important implications in treating chronic pain. However, it is difficult to detect pain level by biotechnology in the early stage of nerve injury in an animal model. Thus, this requires a reliable and steady biochemical marker to evaluate level of pain in animal models. Several markers, such 4.

(18) PK Chao. as c-Fos and microglia were used to detect neuroinflammation and pain levels (Colburn et al., 1997, Munglani et al., 1999, Ro et al., 2004).. 1.3.2 c-fos It is indicated that peripheral tissue injury-induced central sensitization may result from the altered biochemical properties of the spinal dorsal horn (DH) neuron. c-fos is a member of the immediate early gene family and has been used as a tool to study specific neural pathways activated in various animal model (Delander et al., 1997, Li et al., 2004, Huang et al., 2010). In the pain signaling pathways, c-fos is promptly expressed in neurons in response to a repeated touch stimulus (Bester et al., 2000) and a nociceptive stimulus (Sager et al., 1988). Many reports have shown that the expression of c-fos mRNA or protein is up-regulated in the spinal cord following noxious stimulations (Rodella et al., 1998, Wu et al., 2001, Ro et al., 2004, Lee and Seo, 2008). In contrast, expression of c-Fos in the spinal cord was attenuated by pretreatment of analgesia (Lee and Seo, 2008). Thus, expression of c-fos mRNA or protein was used to be a marker of neurons activated by pain sensation (Harris, 1998, Ro et al., 2004).. 1.3.3 microglia In addition to c-Fos, microglia is also either up- or down-regulated in spinal DH. Microglia is the dominant glial cell in the central nervous 5.

(19) PK Chao. system and plays critical roles in neuroinflammation and neuronal plasticity via active communication with neurons (Hanisch and Kettenmann, 2007, Zhuo et al., 2011). In response to peripheral nerve injury,. microglia. become. activated. and. release. a. variety. of. proinflammatory mediators such as cytokines and chemokines in the spinal DH (Inoue and Tsuda, 2009, Gao and Ji, 2010, Hanamsagar et al., 2011). During inflammation process, microglia produced mediators affect on nociceptive neurons, resulting in magnification of nociceptive signal transmission or central sensitization. In the CNS, prostaglandin E2 (PGE2) is one of the early inflammatory mediators released by microglia. Some studies have identified a putative signaling mechanism between activated microglia and pain processing neurons whereby PGE2 is a central molecule in microglia-mediated chronic pain (Zhao et al., 2007). In contrast, modulating microglia function appears to represent a promising therapeutic strategy for neuropathic pain (Muscoli et al., 2010). Therefore, microglia activation could modulate other glial cell function and could be a nociceptive marker (Stuesse et al., 2000, Tanga et al., 2004). Because unilateral nerve injury may have a contralateral effect, Ro et al. (Ro et al., 2004) have showed that the contralateral side of chronic constriction injury (CCI) and sham rats had a greater number of Fos-like immunoreactive neurons compared to that of normal control rats at different time points. Interestingly, although the contralateral side of CCI and sham rats showed a significant increase of the number of Fos-like immunoreactive neurons compared to that of normal control rats, their contralateral hindlimbs did not show evidence of thermal hyperalgesia. 6.

(20) PK Chao. This indicated that c-Fos protein expression has been activated in the spinal DH neurons but did not correlate with nociceptive stimulus induced thermal hyperalgasia in the early inflammation stage after peripheral nerve injury. Our aims are to further investigate whether c-Fos and/or microglia will synchronize with the nociceptive behaviors in the acute phase of nerve injury, we focus on bilateral spinal cord c-Fos protein expression and microglia activation and its relationship to mechanical allodynia in a rat neuropathic pain model.. 7.

(21) PK Chao. 1.4 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 8.

(22) PK Chao. 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 9.

(23) PK Chao. 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 10.

(24) PK Chao. 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 11.

(25) PK Chao. 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.. 12.

(26) PK Chao. 2. Methods. 2.1 Experimental animals 2.1.1 Subjects Adult male Sprague–Dawley rats (BioLASCO Taiwan Co., Ltd. Taipei, Taiwan) weighing around 200–250 g were used. The animal room was artificially lit from 6:00 h to 18:00 h. Three rats were housed in each cage in a temperature-controlled (24°C) animal colony; pellets of rat chow and water were available ad libitum. All behavioral procedures took place during the light cycle. All procedures were conducted in accordance with the Guidelines for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee (IACUC) at the National Taiwan Normal University. Every effort was made to minimize animal suffering as well as the number of animals required to generate reliable experimental data. In TBI model, adult male Wistar rats weighing 350 to 400 g were used in the current study. The rats were also purchased from BioLASCO, Taiwan, Co., Ltd. All animal experiment protocols were approved by the IACUC of the National Chia-Yi University.. 2.1.2 Surgical procedures All experimental procedures were performed on rats that were deeply anesthetized with sodium pentobarbital (50 mg/kg body weight, intraperitoneal (i.p.) injection). Sterile operating instruments were used. Briefly, in CCI rat, 4 ligatures of 4-0 chromic gut were loosely tied 13.

(27) PK Chao. around the nerve with approximately 1.0–1.5 mm interval between the knots such that epineural circulation was preserved. At the time of tying, the ligatures just barely reduced the nerve diameter (Bennett and Xie, 1988). Sham operations were also carried out under the effect of anesthesia to expose, but not manipulate, the right sciatic nerve. The left sides of all rats were not subjected to any surgery. As a TBI model, a special weight-drop device which contained a foam bed on the bottom similar to that described by Marmarou et al. (Marmarou et al., 1994) was used to deliver a standard traumatic impact to the animals. A midline incision was made on the head with a scalpel, and the skin flaps around the cutting site were peeled off laterally. After this, a metal helmet was sewn onto the top of the skull to prevent fracture from the trauma-inducing impact. Rats were then placed in a prone position on the bottom plate of the weight-drop device, and a 450-g weight was allowed to fall freely and vertically from a height of 1.8 m onto the metal helmet to induce TBI.. 2.1.3 Protocols of drug treatments Postoperative intravenous (i.v.) G-CSF injections (200 μg/kg Figrastim; Kyowa Hakko Kirin, Japan) were given to examine the effect on neuropathic pain. Six rats received CCI plus an immediate single i.v. G-CSF injection and 6 received CCI plus an immediate single i.v. vehicle (normal saline) injection. 24 rats were used to examine whether G-CSF alters pain thresholds in sham and/or naïve animals; 6 (12 total) each received a single injection of G-CSF (naïve or sham + G-CSF, respectively) and the same number received a single injection of vehicle 14.

(28) PK Chao. (naïve or sham + vehicle, respectively). 36 rats were used to examine the effect of G-CSF on neuropathic pain responses and determine its therapeutic window; each of the 6 rats received CCI plus i.v. G-CSF or vehicle 24, 48, and 72 h after operation. To examine the counteractive effects of naloxone methiodide (NLXM; Sigma, St. Louis, MO) in G-CSF-induced antinociception experiments, G-CSF was injected alone or with NLXM in each of the 6 rats. Three days after CCI, rats under ether anesthesia received NLXM (30 μg) in a total volume of 180 μL at the same site of nerve injury. A polyethylene tube was placed 1 cm from the tip around a 26 G needle to ensure the same depth of needle insertion into the middle of the scar tissue after CCI. The injection site was reproducibly covered by approximately 1 cm of the nerve, including the ligation site and sites proximal and distal to it (Labuz et al., 2009).In the experiments studying drug effects on the expression of Nogo-A and traumatic brain injury-associated inflammation and axonal damage, the rats were injected with. Nogo-A. antisense. oligonucleotide. (5′-TGCTTTCGGTTGCTGAGGTA-3′) (i.c.v., 5 μl) (Zhang et al., 2007) or indomethacin (i.p., 2.5 mg/kg, dissolved in 75% alcohol, Sigma, St. Louis, Missouri, USA), which compare to be injected with Nogo-A irrelevant control oligonucleotide (5′-GCAGACCAGCGCGGAGCT-3′) or solvent vehicle (75% alcohol) at the time of surgery while anesthetized.. 15.

(29) PK Chao. 2.2 Behavioral tests 2.2.1 Habituation: Prior to testing, each animal was placed for a 10 min habituation period in a test box with the dimensions of 30 × 30 × 15 cm3 having 3 mirrored sides to minimize stress. No food or water was available to the rats during the experiment. Each animal was used only once and was euthanized at the end of the experiment by administering a lethal dose of pentobarbital (Wang et al., 2010).. 2.2.2 Thermal hyperalgesia test: To avoid bias, surgery and behavioral testing were performed by 2 different investigators. CCI-induced thermal hyperalgesia was measured by the latency of hind paw withdrawal from a hot water bath (YIHDER Water Bath BH-230) (46°C) stimulus. The rat was gently held in a towel and the hind paw immersed in hot water. Paw-withdrawal values were obtained before and after the surgical operation and intravenous injections of G-CSF or normal saline. A cut-off time of 20 s was imposed to avoid tissue injury. The average of 3 readings (allowing 10 min intervals between paw withdrawals to prevent sensitization) for paw withdrawal was calculated.. 2.2.3 Mechanical allodynia test: Mechanical allodynia was assessed by von Frey hair, according to a previously described protocol (Zhang et al., 2009). von Frey hairs were applied to the central region of the plantar surface of a hind paw in ascending order of force (0.7, 1.2, 1.5, 2.0, 3.6, 5.5, 8.5, 11.7, 15.1, and 16.

(30) PK Chao. 29 g). Each filament was applied 5 times. When the rats showed 1 withdrawal response to a given filament, the bending force of that filament was defined as the mechanical threshold intensity. The median threshold intensity was calculated from the values following 1 descending and 2 ascending trials. The experimental conditions were identical for both naïve and experimental rats. Behavioral testing resumed a day after the operations and continued for 25 consecutive days.. 2.3 Blood cell counting and flow cytometry For cell counting, we used K3 EDTA VACUTAINER blood collection tubes (BD Biosciences, Heidelberg, Germany) to collect heart blood from deeply anesthetized rats (n = 6 rats per group) and analyzed with a semi-automated hematology analyzer (SYSMEX F820). Cell suspensions were prepared from the surgical sites and stained as follows: tissue fluid (2 days after CCI, 100 µL) was collected from deeply anesthetized rats, fixed and lysed as described by the FACS lysing solution® kit (BD Biosciences), and pressed through a 70 mm nylon filter. For intracellular stains, the cells were fixed with 1% paraformaldehyde and permeabilized with saponin buffer (0.5% saponin, 0.5% bovine serum albumin, and 0.05% NaN3 in phosphate-buffered saline (PBS); Sigma). Permeabilized cells were incubated with different primary antibodies, and the samples were stained using mouse anti-rat CD45 -phycoerythrin (PE) cyanine dye 5, and incubated with a conjugated polyclonal antibody (CD45; 1:500; BD PharMingen, CA) for labeling all leucocytes.. To. differentiate. the 17. leukocyte. subpopulations,. cell.

(31) PK Chao. suspensions. were. stained. with. fluorescein. isothiocyanate. (FITC)-conjugated rabbit anti-rat polyclonal antibody recognizing granulocytes (PMN; 1:500; Accurate Chemical, USA) and PE-conjugated mouse anti-rat macrophages monoclonal antibody (ED2; 1:500; AbD Serotec, United Kingdom). For intracellular staining, the cells were prepared and incubated with rabbit anti-rat β-endorphin-PE polyclonal antibody that can recognize opioid peptides (β-endorphin; 1:500; Bioss). The specificity of the staining was verified by incubating cell suspensions with the appropriate isotype-matched control antibodies. To calculate the absolute number of PMN cells around the stained cell suspension, 10,000 fluorescence-activated cell sorting (FACS) events were acquired. All obtained data were analyzed using CellQuest software (BD Biosciences, USA).. 2.4 IL-6 mRNA assay 2.4.1 RNA extraction and reverse transcription PCR (RT-PCR): Lumbar 5 (L5) dorsal root ganglia (DRG) were excised from naïve rats and rats with or without G-CSF treatment (n = 5 per group) 3, 6, 12, 24, 48, 72 and 144 h after CCI. L5 DRG samples were frozen in liquid nitrogen, and then ground to a fine powder by using a mortar and pestle. The total RNA of L5 DRG was extracted with TRIzol® reagent, according to the manufacturer’s instructions. RNA was converted into cDNA using a first strand cDNA synthesis kit (cat. 18080-051; Invitrogen) with 18.

(32) PK Chao. random hexamers.. 2.4.2 Quantitative RT-PCR: Quantitative RT-PCR was carried out in a final volume of 25 μL comprising optimal concentrations of primers and probes in 96-well plates on an ABI PRISM 7000 detector (Applied Biosystems, Foster City, CA). The primers and probes were purchased from Applied Biosystems and delivered as 20X concentrates. The reaction mixture for RT-PCR comprised 12.5 μL of Taqman® Universal PCR Master Mix containing 1.25 U of AmpliTaq DNA polymerase, 1.25 μL of probes, 1–2 μg of L5 DRG cDNA, and water to a final volume of 25 μL. TaqMan® rat IL-6 MGB probes (cat. Rn99999011_m1) were labeled with a FAM reporter fluorescent dye and TaqMan® rat GAPDH MGB probes (P/N 4352338E), with a VIC reporter fluorescent dye. The amplification started with 2 min at 50°C, 10 min at 95°C, followed by 50 cycles of the following: 15 s at 95°C and 1 min at 60°C. Amplicons were run in di-duplicate in a separate tube assay for the quantification of IL-6 gene in comparison to the assays for the internal control gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ΔCt represents the mean Ct value of each sample and was calculated for both IL-6 and GAPDH. The relative fold change of IL-6 expression in naïve rats in L5 DRG was calculated by the comparative Ct method (2 -(ΔΔCt)) of relative quantification.. 19.

(33) PK Chao. 2.5 Western blotting assay Sixteen rats were subjected to CCI and received an immediate single i.v. G-CSF injection, 16 were subjected to CCI and received vehicle injection, and 16 underwent surgery without CCI and received vehicle injection. Four rats from each group were killed at 24, 48, 72, and 144 h after operation. In addition, the post-TBI rats (n = 6 in each group) were decapitated and the brains were removed at different time points after TBI. The L5 DRG, L6 DRG, L4 to L6 segments of spinal cord, and hippocampus were resected and briefly sonicated in ice-cold buffer (50 mM Tris-HCl [pH 7.8], 50 mM NaCl, 10 mM ethylene glycol tetraacetic acid [EGTA], 5 mM EDTA, 2 mM sodium pyrophosphate, 4 mM para-nitrophenylphosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSP), 20 ng/mL leupeptin, and 4 ng/mL aprotinin). The samples were ultracentrifuged at 13,600 rpm for 30 min at 4°C. Bradford assay was performed with the lysates to ensure that an equal quantity of protein was loaded into each well. Before the western blotting assay, equal protein loading was verified using Coomassie staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein samples were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The blots were blocked using 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.5% Tween-20 overnight at 4°C and then incubated with TNF-α (1:2000; Millipore, Billerica, MA, USA) or GAPDH (internal control; 1:2500; Alpha Diagnostic 20.

(34) PK Chao. International, San Antonio, TX, USA) primary antibody in TBS containing 1% BSA and 0.5% Tween-20 overnight at 4°C. This step was followed by incubation with horseradish peroxide (HRP)-linked secondary antibody (1:2500, anti-rabbit IgG; Cell Signaling Technology) for 45 min at room temperature. All washing was performed using TBS containing 0.1% Tween-20. The bands were detected using an enhanced chemiluminescence western blotting analysis system (RPN 2108; Amersham International, Amersham, UK).. 2.6 Enzyme-linked immunosorbent assay (ELISA) The L5 samples of the experimental group were subsequently analyzed in duplicates in a double-blinded manner. TNF-α levels in the soluble fraction of DRG were measured by ELISA using a commercially available kit, according to the manufacturer instructions (Biosource/Invitrogen, Carlsbad, CA, USA). The concentration of IL-1 was also measured using a commercial ELISA kit according to the manufacturer’s instructions (Bender Medsystems, San Diego, CA, USA).. 2.7 Immunohistochemistry Rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with PBS (Sigma), followed by a fixative solution containing 4% paraformaldehyde. The spinal cord segments (L4-L6) and sciatic nerves were resected and placed in 4% 21.

(35) PK Chao. paraformaldehyde for 4 h and transferred to 30% sucrose at 4°C overnight. The samples were subsequently embedded in OCT compound (Tissue-Tek 4583; Sakura, Tokyo, Japan) and rapidly frozen. Tissue sections of 10 μm thickness were obtained using a freezing microtome (CM 3050; Leica, Nussloch, Germany), mounted on polylysine-coated slides. After rinsing with cold PBS (0.1 M, pH 7.4), the sections were heated in citrate buffer for 20 min at 90°C for antigen retrieval.. In. microglial. response. states,. after. incubation. of. protein-blocking buffer (BioGenex, San Ramon, CA) for 1 h at room temperature, the sections were incubated at 4°C overnight with a mouse anti-rat CD11B primary antibody (OX-42 clone, 1:1000 dilution; Millipore). After rinsing, the sections were assayed with the Super Sensitive Polymer-HRP IHC Detection System (BioGenex), according to the manufacturer’s instructions. Peroxidase activity was developed by. treatment. with. 3,3′-diaminobenzidine. for. 2. min.. In. immunofluorescence analysis, the sections were blocked with protein-blocking buffer (BioGenex, San Ramon, CA) and incubated with rabbit anti-β-endorphin antibody (1:200; Chemicon, Neuromics, Minneapolis, MN) for 24 h at 4°C, followed by the secondary antibody (1:200, TRITC-conjugated goat anti-rabbit IgG; Jackson Immuno Research Laboratories, Inc., USA) for 1 h at room temperature. After β-endorphin immunostaining, the sections were further processed with PMN immunostaining with a rabbit anti-rat PMN-FITC antibody (1:200; Accurate Chemical, NY) (Nguyen et al., 2008) for 2 h at room temperature. The Images were acquired using a confocal spectral 22.

(36) PK Chao. microscope (Leica TCS-SP2, Heidelberg, Germany) connected to digital camera and computer; montages were created and analyzed using Image-ProPlus 5.0 (Media Cybernetics Inc., Silver Spring, MD). The areas occupied by the stained tissues were highlighted and measured for each section of the sciatic nerve (n = 5 per group, 5 sections per animal) and expressed in terms of the density (number/μm2) of the right sciatic nerve. Omission of the primary antibody served as the negative control for all experiments.. 2.8 Statistical analysis All statistical comparisons were made using SPSS version 15.0 (SPSS Inc., Chicago, IL). Normally distributed data were analyzed by Student's t-test (for 2 groups) or one-way analysis of variance (ANOVA) (for 3 or more groups). If the data lacked equal variance or normality, Mann–Whitney rank sum test or Kruskal–Wallis test was used. Data derived from behavioral experiments were analyzed by repeated measures two-way ANOVA across testing time points to detect overall differences across different treatment groups. In both cases, when significant main effects were detected, post hoc Newman–Keuls test was performed to determine the source of the difference. Data are expressed as mean ± standard error of the mean. A p value less than 0.05 was considered significant.. 23.

(37) PK Chao. 3. Results. 3.1 Part I. 3.1.1 The nociceptive response of mechanical stimuli is enhanced in the early stage of CCI rats. The sham rats exhibited normal mechanical allodynia compared to the CCI rats at 48 and 72 h after operation. In contrast, after unilateral CCI of the sciatic nerve, animals developed significant mechanical allodynia on ipsilateral side at 48 and 72 h (Fig. 1; *p < 0.05). Interestingly, to compare with sham, CCI rats notably developed mechanical allodynia on contralateral side in a less degree at 48 and 72 h (Fig. 1; #p < 0.05). Furthermore, the nociceptive behaviors of contralateral side slightly synchronize but significantly different from ipsilateral side (Fig. 1; $p < 0.05). In contrast, sham surgery did not alter the mechanical allodynia in sham-operated rats in both ipsilateral and contralateral sides (Fig. 1). Thus, tissue injury without nerve injury did not induce significant mechanical response and central sensitization, suggesting that bilateral mechanical allodynia development may be dependent on nerve injury. Moreover, levels of mechanical allodynia on ipsilateral sides were higher than those on contralateral sides at 48 and 72 h, respectively.. 24.

(38) PK Chao. 3.1.2 The immune cells distribute in the sciatic nerve. During nerve injury, pain may be associated with migration of immune cells and release of pro-inflammatory cytokines (Hu et al., 2007, Grace et al., 2011). In our study, nerve sections from the sciatic nerves were obtained at 72 h after CCI and increased number of immune cells [stained with mouse anti-CD45 (common leukocyte antigen) antibody] was observed only in the injury side (Fig. 2B) but not in the contralateral side (Fig. 2A).. 3.1.3 Expression of c-Fos protein distribute in the bilateral DRG and spinal DH in the early stage after CCI There was no difference in the levels of c-Fos protein between the sham-operated and CCI-operated rats in the contralateral DRGs (Fig. 3). Interestingly, western blotting assay was performed at 72 h after the operation showed that the c-Fos protein expression in the ipsilateral DRG was not increased by CCI treatment (Fig. 3). Furthermore, we examined the levels of c-Fos protein in bilateral spinal DH of sham-operated and CCI rats at 72 h. We found the levels of c-Fos protein was only increased in the ipsilateral spinal DH of CCI rat (Fig. 4; *p < 0.05). It is suggesting that the level of c-Fos protein was up-regulated in the CNS, but unchanged in PNS after unilateral nerve injury.. 25.

(39) PK Chao. 3.1.4 Microglia activation distributes in the spinal DH in the early period of CCI rats In separate groups of animals, sections obtained from the spinal cord were stained with OX-42 to detect activated microglia. The expression patterns of OX-42 immunoreactivity in the spinal DH of sham-operation and CCI-operation rats were compared at 72 h after CCI. In CCI rats, the microglial cells were markedly activated in both of the ipsi- and contra-lateral spinal DH at 72 h after surgery. In contrast, sham rats exhibited significantly lower levels of OX-42 immunoreactivity in the bilateral spinal DH compared to the CCI rats (Fig. 5A). The increased number of activated microglia was noted in the superficial and deep DH bilaterally (Fig. 5B; ipsilateral: *p < 0.05, contralateral: #p < 0.05). In addition, ipsilateral number of OX-42 positive immunoreactivity of the superficial and deep spinal DH were higher than that of contralateral side in CCI rats (Fig. 5B; $p < 0.05).. 3.1.5 Correlation between mechanical paw withdrawal response and microglial activation We found the number of OX-42 positive immunoreactivity is significantly correlated with mechanical paw withdrawal responses in different laminae of DH at 72 h. At L5 segment, the number of OX-42 positive immunoreactivity in superficial (r=−0.837; p < 0.05) and deep (r=−0.998; p < 0.05) spinal DH is negatively correlated with mechanical paw withdrawal response in CCI rats (Fig. 6). In contrast, there was no 26.

(40) PK Chao. correlation between microglial activation and mechanical paw withdrawal response in sham rats. However, there is no significant correlation between the paw withdrawal response and the number of OX-42 immunoreactive cells at the superficial or deep spinal DH in sham rats.. 3.1.6 Correlation of microglial activation between ipsi- and contra-lateral or superficial and deep spinal DH Furthermore, the number of OX-42 positive immunoreactivity at the deep spinal DH were correlated with superfical side (r=−0.764; p < 0.05) (Fig. 7B). Similarly, OX-42 immunoreactivity levels at the contralateral spinal DH were correlated with ipsilateral side (r=−0.796; p < 0.05) (Fig. 7A). However, in sham rats, there is no significant correlation between the number of OX-42 immunoreactive cells at the superficial and deep spinal DH and there is no correlation between the ipsi- and contra-lateral spinal DH either. These results indicate that the a significant increase the number of activated microglia at ipsilateral spinal DH may affect the contralateral spinal DH, which will decrease the threshold of mechanical paw withdrawal response in CCI rats and will induce further central sensitization and develop the neuropathic pain.. 27.

(41) PK Chao. 3.2 Part II. After unilateral CCI of the sciatic nerve, animals developed thermal hyperalgesia (Fig. 8A; from day 2 to day 25) and mechanical allodynia (Fig. 8B; from day 3 to day 25) of the right injured hindpaw compared to naïve (two-way repeated-measures ANOVA, p < 0.01) or sham-operated rats (two-way repeated-measures ANOVA, p < 0.05). Post-CCI thermal hyperalgesia and mechanical allodynia were not altered by the infusion of the vehicle (saline); in contrast, CCI rats with i.v. injection of G-CSF exhibited significantly attenuated thermal hyperalgesia and mechanical allodynia compared to the rats treated with the vehicle (p < 0.01) (Fig. 8A, B). However, no significant alterations in thermal or mechanical sensations were observed when G-CSF was injected into sham-operated animals (Fig. 8C, D). A lack of effects on thermal and mechanical sensations was observed in naïve animals who received either G-CSF or vehicle (Fig. 8E, F).. 3.2.1 G-CSF affects the nociceptive behaviors in rats after nerve injury. To determine the therapeutic window of G-CSF and to determine whether G-CSF is effective in reversing thermal hyperalgesia once established, delayed G-CSF injections were administered 24, 48, and 72 h after CCI. Delayed G-CSF injections (24 and 48 h after CCI) resulted in a significant reversal of thermal hyperalgesia compared to the effect of only 28.

(42) PK Chao. vehicle treatment (p < 0.05) (Fig. 9A, B). These findings are similar to those in rats with immediate G-CSF injections, which significantly attenuated thermal hyperalgesia. However, delayed G-CSF injections 72 h after CCI did not significantly attenuate thermal hyperalgesia (data not shown). Thus, the effect of G-CSF on the attenuation of thermal hyperalgesia of CCI rats has a therapeutic window of 0–48 h after nerve injury.. 3.2.2 Effects of G-CSF on mobilized bone marrow cells in the peripheral blood and tissue fluid around the injured nerves. Cell counting analysis of immune cell subpopulations in peripheral blood showed that the count of specific immune cells significantly increased in CCI rats treated with G-CSF compared to those that received vehicle treatment (Fig. 10). At 3 to 48 h after nerve injury, CCI rats treated with G-CSF exhibited increased total counts of WBCs (1.72- to 2.03-fold) (Fig. 10A) and PMN cells (1.86- to 2.14-fold) (Fig. 10B) in the peripheral blood compared to those treated with vehicle only. The count of circulating lymphocytes and monocytes increased but not significantly (data not show). Multi-color flow cytometry, was used to examine opioid peptide expression and expression of PMN cell markers. On comparing the extent of migration of immune cells (CD45 marker cells) into the tissue fluid (Fig. 11) around the injury sites in G-CSF- and vehicle-treated CCI rats, we found that the number of opioid containing PMN cells in the G-CSF-treated CCI rats was greater than that in the vehicle-treated CCI 29.

(43) PK Chao. rats (Fig. 11A). To clarify the specificity of the PMN cell marker, we used ED2-PE to stain immune cells of the tissue fluid around the injured nerves. We found only a negligible amount of cells showing ED2-PE and PMN-FITC co-staining, indicating anti-PMN-FITC antibody is specific for PMN labeling. Furthermore, the percentages of opioid-containing leukocytes were significantly different in the tissue fluids between the 2 groups (Fig. 11B; number of opioid-positive cells in the total PMN cells, vehicle control: 55.8% ± 9.4% and G-CSF: 79.2% ± 3.5%; statistical analysis by t test, p < 0.05). The animals without inflammation (naïve) were not analyzed because of lack of tissue fluid in the nerve tissue.. 3.2.3 Effects of G-CSF associate with the peripheral opioid/opioid receptor interaction in injured nerves. Nerve sections from the sciatic nerves were obtained at 3, 6, 12, 24, 48, and 72 h after CCI and were double stained with polyclonal anti-β-endorphin. and. anti-PMN. cell. antibodies.. The. immunohistochemical photomicrographs revealed that the majority of PMN cells also expressed β-endorphin, which was more prominent from 12 to 48 h after injury in CCI rats treated with G-CSF than in those treated with the vehicle (Fig. 12A–C). Increased migration of PMN cells was observed only in the injury sites (Fig. 12B-D) but not in the sham-operated nerves (Fig. 12A). After CCI, the number of opioid-containing PMN cells at 12–48 h in G-CSF-treated rats was significantly higher than that in vehicle-treated rats; further, the number 30.

(44) PK Chao. of PMN cells increased earlier in G-CSF-treated rats than in vehicle-treated rats (Fig. 12E). In addition, nerve sections from the sciatic nerves were obtained at 24 h after CCI and were stained with polyclonal anti-μ-opioid receptor antibody. The immunohistochemical photomicrographs revealed that the majority of sciatic nerve expressed μ-opioid receptor, there were immunoreactivity at 24 h after injury in CCI rats treated with vehicle or G-CSF (Fig. 13B,C) and in sham-operated rats treated with vehicle (Fig. 13A). After unilateral CCI of the sciatic nerve, animals developed thermal hyperalgesia and mechanical allodynia. In contrast, CCI rats treated with G-CSF exhibited normal thermal and mechanical responses compared to the CCI rats treated with vehicle 72 h after operation. Interestingly, G-CSF-treated CCI rats rapidly redeveloped thermal hyperalgesia (Fig. 14A; p < 0.05) and mechanical allodynia (Fig. 14B; p < 0.01) after NLXM. injection. at. that. time;. however,. NLXM-regulated. analgesia-blocked effect lasted for a maximum of 1 h, after which the G-CSF-treated CCI rats returned to their original normal thermal and mechanical response state. Thus, the analgesic effects of G-CSF on thermal hyperalgesia and mechanical allodynia are opioid/opioid receptor interaction dependent.. 31.

(45) PK Chao. 3.2.4 Effects of G-CSF on different pro-inflammatory cytokines expression were detected in the bilateral DRGs and spinal DHs after CCI. Pro-inflammatory cytokines, such as IL-1β, IL-6 and TNF-α, have been implicated in the development of hyperalgesia or allodynia after nerve injury. Real-time PCR revealed that the IL-6 mRNA expression in the L5 DRG of the injured sides of CCI rats increased 24 h after injury, and the increase persisted until 144 h after CCI. Compared to vehicle treatment, G-CSF treatment down-regulated IL-6 expression within 48 – 144 h after CCI (Fig. 15A). ELISA showed that TNF-α (Fig. 15B) levels of G-CSF-treated rats increased 24 h after operation, whereas protein expression in the DRG was attenuated at 72 and 144 h after the operation. The peak levels in the vehicle-treated groups were higher than those in the G-CSF-treated groups. There were no differences in the peak levels between. the. sham-operated. group. and. G-CSF-treated. groups.. Furthermore, western blotting assay performed at 72 h after the operation showed that the TNF-α protein expression in the DRG was attenuated by G-CSF treatment (Fig. 15C). However, throughout experimental period, TNF-α protein expression did not vary in the internal control samples.We examined the levels of IL-1β protein in bilateral spinal DH of sham-operated rats, vehicle-treated CCI rat and G-CSF-treated CCI rats at 24 h. We found the levels of IL-1β protein was increased in the ipsilateral spinal DH of vehicle-treated CCI rat (Fig. 16; *p < 0.05).. 32.

(46) PK Chao. 3.2.5 G-CSF affects the microglial activation in rats after nerve injury. Furthermore, in separate groups of animals, sections from the spinal cord obtained 72 h after CCI were stained with OX-42 to detect activated microglia. The expression patterns of OX-42 immunoreactivity in the DH of naïve, sham, vehicle and G-CSF-treated rats were compared (Fig. 17A–D). In CCI rats treated with vehicle, the microglial cells were markedly activated in the ipsilateral DH 72 h after surgery (Fig. 17C). In contrast, CCI rats treated with G-CSF (similar to sham rats) exhibited significantly lower levels of OX-42 immunoreactivity compared to the vehicle-treated rats 72 h after nerve injury (Fig. 17D). Up-regulation of IL-1β protein was observed at 24 h (Fig. 16) and increased activated microglia numbers was noted at 72 h after CCI. Microgliosis was defined as cells displaying hypertrophy of cell bodies, and retraction of processes, with overlap individual microglia. These indicate that expression of pro-inflammatory cytokines, such as IL-1β in the spinal cord DH, IL-6 and TNF-α in the DRG, may trigger the microglial activation and the development of hyperalgesia or allodynia after nerve injury. There is evidence that the activated microglia numbers increased at inflammatory spinal DH after CCI with vehicle treated but attenuated after G-CSF injection (Fig. 17E).. 33.

(47) PK Chao. 3.2.6 Up-regulation of Nogo-A in TBI model. A previous study demonstrated that NgR is associated with microglia in CNS (Satoh et al., 2007), which implies that Nogo-A plays a role of microglial activation induced CNS inflammation. In order to primary detect the role of Nogo-A in neuroinflammation of CNS, the next experiment conducted in the current study sought to examine alterations in the expression of Nogo-A in the hippocampus after TBI. This stimulatory effect on Nogo-A production was confirmed by protein analysis. Western blot analysis revealed an increase in Nogo-A protein in the hippocampus four hours post-TBI. However, a statistically significant elevation in the protein level began at eight hours after TBI and lasted for three days (data not shown). Moreover, this TBI-induced stimulation of Nogo-A expression could be reversed by the administration of Nogo-A antisense oligonucleotide immediately after TBI. As shown in the western blot analysis (data not shown), microinjection of Nogo-A antisense oligonucleotide into the lateral ventricle drastically decreased the TBI-induced Nogo-A production by approximately 70%. However, the Nogo-A irrelevant control oligonucleotide appeared to be ineffective in decreasing the TBI-associated Nogo-A production.. 3.2.7 Indomethacin attenuated expression of Nogo-A The level of Nogo-A was again significantly increased as a consequence of TBI, whereas in the TBI rats that were given indomethacin, Nogo-A expression at the protein (data not shown) levels 34.

(48) PK Chao. returned to those observed in sham animals. Indomethacin, a potent non-steroidal anti-inflammatory drug, was used in this experiment to determine the relationship between TBI-associated inflammatory effects and Nogo-A expression. Unlike the direct effect conferred by Nogo-A antisense oligonucleotide, indomethacin may conceivably have triggered a novel pathway that resulted in the suppression of Nogo-A expression. However, an interesting finding was that the administration of indomethacin or Nogo-A antisense, but not Nogo-A irrelevant control oligonucleotide, not only suppressed the Nogo-A overproduction but also down-regulated the expression of protein (data not shown) after TBI. This strongly suggests that TBI-induced IL-1β production is modulated by the level of Nogo-A. Additionally, as described above, the change in the level of IL-1β is modulated by that of Nogo-A, suggesting that the alteration of Nogo-A expression might be an early stage event in the protection process conferred by indomethacin.. 35.

(49) PK Chao. 4. Discussion. 4.1 Part I To detect the relation between biochemical and phenotypic changes, some markers, such as c-Fos protein expression and microglia activation were widely used in pain research of the CNS (Jergova et al., 2008, Graeber, 2010). However, it has been shown that lower threshold stimuli, such as Aβ fiber activation normally do not induce the expression of c-Fos, which is not correlated with nociceptive behavior at different times. To further investigate whether c-Fos protein and activated microglia are involved in early neuroinflammation and related to nociceptive behaviors, we analyzed the mechanical paw withdrawal response and distribution of both bio-markers in CCI rats. Although unilateral CCI c-Fos protein expression was unchanged in the ipsilateral DRG (Fig. 3) but increased in ipsilateral spinal DH (Fig. 4) at 72 h after nerve injury, animals still developed mechanical allodynia in both ipsi- and contra-lateral limbs (Fig. 1). In contrast, Nerve injury increased activated microglia in both ipsiand contra-lateral spinal DH at 72 h, and the number of activated microglia in the ipsilateral DH was higher than the contralateral side. Moreover, the number of activated microglia at deep spinal DH is well correlated with mechanical paw withdrawal responses. This finding suggests that the number of activated microglia in the deep laminae (III, IV) of spinal DH may decrease the threshold of mechanical response and induce widespread central sensitization; which will establish the 36.

(50) PK Chao. development of neuropathic pain.. 4.1.1 The role of c-Fos in the DRG of CCI rat Interestingly, we found that c-Fos protein was unchanged in the ipsilateral DRG of CCI rat compare to sham rat. Moreover, the level of c-Fos protein in the ipsilateral DRG of CCI rat was slight down-regulated than the contralateral side of sham-orerated rat (Fig 3). This implied that there are some inhibiting mechanisms involved in c-Fos protein expression in injured nerve in the early stage. The current studies showed that reduction of c-Fos protein expression was found after administration of analgesic drugs. For example, nociception-induced c-Fos protein expression in spinal DH neurons is suppressed by local administration of analgesic drugs including morphine (Gogas et al., 1991, Jasmin et al., 1994) and indomethacin (Honore et al., 1995). In our study, we found that immune cells infiltrate into the peripheral injured nerve (Fig 2). As compared with previous flow cytometry study, 30–40% of the migrating CD45+ cell express opioid peptides in the injured sciatic nerve, which will suppress the nociceptive signals transmission (Labuz et al., 2009). In addition, the number of opioid-containing cells in the injured nerve of CCI rats was higher than those of sham CCI-operated compared to sham-operated nerves (Labuz et al., 2009). Similar anti-nociceptive effects produced by immune cell derived opioid peptides interact with opioid receptors at the nerve injury site in CCI model (Labuz et al., 2009). Inhibition of calcium channels should be a major mechanism of 37.

(51) PK Chao. opioid receptor function in the DRG (Akins and McCleskey, 1993). Moreover, the mechanisms of opioid receptor include blocking sodium channels (Gold and Levine, 1996) and TRPV channels (Endres-Becker et al., 2007), which showed that opioids could attenuate the excitability of DRG neurons. Additionally, increase of μ- and δ-receptor proteins at the nerve injury site was detected by Western blot in the CCI models (Truong et al., 2003). Immunoreactivity of μ-receptor was increased distally to the injury at 2 and 14 days in CCI rat compared to intact nerves (Truong et al., 2003). Importantly, these opioid receptors of nerves and the DRGs are functional, because their activation following exogenous opioid application result in the attenuation of mechanical allodynia and thermal hyperalgesia (Przewlocki and Przewlocka, 2005). This suggests that the migrated immune cells may release opioids to affect opioid receptors of injury site, which partly attenuate excitation of peripheral nociceptive neurons and reduce expression of c-Fos protein in the DRGs.. 4.1.2 c-Fos expression of the spinal DH and pain-related behaviors Since there are several major types of sensory fiber in peripheral nerves including myelinated Aβ fibers, thinly myelinated Aδ fibers and unmyelinated C fibers, this is difficult to separate the mechanisms of c-Fos protein expression between thermal hyperalgasia and mechanical allodynia from CCI model. But noxious stimuli are transduced into spinal DH through Aδ and C fiber, it suggests that specific receptors and ion 38.

(52) PK Chao. channels locate at the terminal of nociceptive fiber, which transmit nociceptive signals to activate c-Fos protein expression in spinal DH. Transient receptor potential vanilloid (TRPV) ion channels are associated with thermal hyperalgasia, these include TRPV type I, II, and III receptors in nociptors. There were higer staining intensity of Fos-like immunoreactive neurons within ipsilateral spinal DH of rat treated by unilateral capsaicin injection (Wu et al., 2001). Additionlly, electrical stimulation of the sciatic nerve at Aδ or C fiber intensity leads to post-translational modification and expression of transcription factors in superficial spinal DH, which including c-fos mRNA and protein (Herdegen et al., 1991a, Herdegen et al., 1991b, Ji et al., 1999). It has been shown that lower threshold stimuli, such as touch, warm stimuli or Aβ fiber activation could not induce the expression of c-fos. This indicates that stimulus of Aδ and C fiber may activate TRPV ion channels to enhance c-Fos protein expression in superficial spinal DH neurons, and c-Fos protein expression of the spinal DH correlates with thermal hyperalgesia behavior in the early stage of CCI rat (Ro et al., 2004). However, Ro et al. showed a transient increase of the number of Fos-like immunoreactive neurons on both sides of the sham-operated rats compared to naïve rats on post- operation day 2. This indicates that neurons of the spinal DH were activated in the early stage after sham operation without nerve injury. But, neither the ipsi- nor contra-lateral limbs of sham rats showed thermal hyperalgesia (Ro et al., 2004). After sham-operation, a transient increase of c-Fos protein expression may not need a threshold to induce nociceptive behaviors in the early stage. 39.

(53) PK Chao. A brief electrical stimulation of Aδ and C fiber of the sciatic nerve leads to enhance the expression of c-Fos in the superficial spinal DH (Herdegen et al., 1991a, Bullitt et al., 1992). However, a longer electrical stimulation of the sciatic nerve leads to expression of c-Fos in the deep spinal DH (Bullitt et al., 1992). Unilateral capsaicin injection will induce a few numbers of Fos-like immunoreactive neurons within contralateral spinal DH (Wu et al., 2001). Somehow c-Fos protein expression could extend to another ipsilateral laminae (I, V) layer and even to contralateral side of the spinal cord, which are not innervated by Aδ or C fibers. The number of Fos-like immunoreactive neurons on contralateral sides of the CCI-operated rats showed a transient increase at day 2, and then down-regulated to basic level at day 10 (Ro et al., 2004). Similarly, we found that c-Fos protein level of the contralateral spinal DH and in CCI rats was not significantly different from sham-operated rats at day 3. Instead, the lower threshold of mechanical responses did not closely correlate with increase of c-Fos protein expression in spinal DH in the early stage. This suggests that c-Fos protein expression of contralateral spinal DH neuron in CCI rats is a transient increase and does not reach a threshold to induce mechanical allodynia or thermal hyperalgesia.. 4.1.3 Correlation between microglia and pain-related behavior c-fos could be used as a tool to detect neural activation which 40.

(54) PK Chao. induced by various animal models, however, neurons are not the only cells playing a role in neuropathic pain. There is evidence that glia cells (such as astrocyte and microglia) are involved as well, and there is a cross effect between neuron and microglia in the CNS which associated to nociceptive behaviors (Jurgens and Johnson, 2012). In our results, bilateral spinal DH showed an increase of activated microglia; moreover, the number of activated microglia of ipsilateral DH are higher than that of contralateral side (Fig. 5), which correlates with mechanical allodynia behavior in the early stage in CCI rat (Fig. 6). Thus, there are several hypotheses, which may account for the above phenomena. First, peripheral nerve injury often leads to inflammatory reactions which mobilize the central glia activation in the spinal cord. It is demonstrated that a sickness syndrome of peripheral immune system leads to the production of histamine and pro-inflammatory cytokines (Marchand et al., 2005). Injury-induced histamine additional counts of neutrophils and macrophages at the site of nerve damage and enhanced mechanical and thermal hypersensitivity. Additionally, intracutaneous application of histamine enhanced spontaneous pain in patients with postherpetic neuralgia (Baron et al., 2001). Instead, intraperitoneal injections of histamine receptor 4 antagonist reversed mechanical allodynia following CCI (Hsieh et al., 2010). This suggests that immune cells contribute to neuropathic pain by the release of histamine and by the recruitment of other immune cells secreting mediators. Furthermore, exogenous or peripheral cytokines, such as interleukin-1β, interleukin-6, and TNF-alpha, could trigger the synthesis of pro-inflammatory cytokines 41.