Discussion - 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

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 ipsi- and 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

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

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

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.

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

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

by microglia within the central nervous system including the spinal cord (McMahon et al., 2005). In our results, migration of immune cells was observed in the injured side but was not in the contralateral side (Fig. 5).

However, bilateral spinal DH appeared an increase of microglia activation. These unilateral CCI-induced immune cells produced histamine and cytokines possibly induce a global effect to increase contralateral microglia activation on spinal DH in the early stage. In summary, histamine and pro-inflammatory cytokines derived from inflammatory tissues and immune cells, it may induce global inflammation to result in central sensitization after CCI surgery.

Secondary, we found the number of activated microglia in ipsilateral DH was higher than contralateral side in the early stage of CCI rat (Fig.

5). Because there is evidence that most stimuli that lead to microglia activation can cause nociceptive behaviors in animals, spinal microglia may be activated by mediators released from primary afferent nerves and/or spinal DH neurons, such as substance P, pro-inflammatory cytokines, and excitatory amino acids, which may induce mechanical allodynia in the early stage (Gosselin et al., 2010).

Third, during nerve injury, pain is associated with microglia released pro-inflammatory cytokines such as interleukin-1β, interleukin-6, and TNF-alpha (Inoue and Tsuda, 2009). In addition, cytokines produced in ipsilateral spinal DH may expand to contralateral spinal DH to affect biochemical synthesis and nociceptive response in the early stage of CCI model (Jancalek et al., 2010). Instead, functionally active neurons release a number of soluble mediators including neurotransmitters and

neurotrophic factors that keep microglia in a resting state (Jurgens and Johnson, 2012). These suggest that there is a cross talk between neuron and microglia in the ipsilateral spinal DH which may spread the interaction to the contralateral side, which, in turn , induces the nociceptive behaviors. Similarly, in our results, CCI rats notably developed mechanical allodynia on bilateral sides (Fig. 1), as well as nociceptive behavior, microglial cells were markedly activated in the bilateral spinal DH at 72 h after CCI. In addition, the number of activated microglia at superficial and deep laminae in contralateral DH were synchronically increase with but in a less degree ipsilateral side (Fig. 5, 6), which implies that there are pro-inflammatory mediators or excitatory signals that expand from the ipsilateral spinal DH to contralateral side.

We found microglia activation of ipsilateral spinal DH as well as mechanical allodynia of ipsilateral limb was more than contralateral side in CCI rats. This may imply a mild and transient effect on the contralateral spinal DH, which result from the influence of ipsilateral microglia produced pro-inflammatory cytokines after peripheral nerve injury.

4.1.4 Summery

Intense noxious stimulation has been demonstrated to sensitize CNS neurons. It is important for pain research to detect a cell marker for nociception or inflammation induced neuronal activation and central sensitization. c-Fos is a good marker for nociception-induced neuronal activation in the nerve system. However, microglia activation of the

spinal DH is directly related with central sensitization, and closely correlated with mechanical allodynia in early inflammatory stage.

Moreover, it also can be easily detected and quantified by immunohistochemistry. Thus, to detect microglia activation should be a better tool for pain research. This indicates that the microglia activation play a key role of central sensitization.

4.2 Part II

The major finding of the present study is that exogenously applied G-CSF is effective in alleviating thermal hyperalgesia and mechanical allodynia in rats with CCI mainly through the activation of leukocyte-derived endogenous opioid secretion, down-regulation of IL-6 and TNF- inflammatory cytokines, and decreased microglial cell activation in the spinal DH. Prolonged inflammation increases the number of opioid-containing immune cells, tissue endorphin contents, and the efficacy of pain control (Rittner et al., 2001, Mousa et al., 2002, Machelska et al., 2003, Hagiwara et al., 2008, Labuz et al., 2009).

4.2.1 A novel role of G-CSF in antinociception

Our study indicates that G-CSF is a safe drug for antinociception.

Firstly, endogenous G-CSF is usually generated at the site of inflammation and acts as an endocrine hormone to mobilize immune cells from the bone marrow to replace the inflammatory cells consumed in an inflammatory reaction (Becker et al., 1997). In G-CSF receptor knockout mice, the G-CSF-stimulated mobilization of neutrophils and hematopoietic progenitors from the bone marrow to the blood was markedly impaired (Semerad et al., 1999). Thus, G-CSF is a unique and specific receptor-dependent cytokine that drives proliferation of certain specialized hematopoietic cells, and survival and neutrophilic differentiation of myeloid progenitor cells (Takemoto et al., 2000, Touw

and van de Geijn, 2007, Eyles et al., 2008). Secondly, PMN cells become activated during early inflammation and are recruited by the up-regulation of corticotropin-releasing factor (CRF) and chemokines of the CXC family (Brack et al., 2004b), which are secreted by inflamed tissues and immune cells and may be the source of peripheral (or endogenic) endorphin (Brack et al., 2004a). Cell counting revealed that about 12 h after operation, G-CSF increases the number of PMN cells by a larger amount and in a faster manner in blood circulation and injured nerves compared to vehicle treatment. The result indicates that G-CSF may exhibit the powerful function of recruiting PMN cells while not affecting other hematopoietic cells during the early stage of inflammation (Fig. 10). Furthermore, there is no evidence that PMN cells secrete large amounts of β-endorphin in normal circulation, suggesting that PMN cells only secrete β-endorphin under certain circumstances. Moreover, G-CSF does not affect nociceptive responses in naïve and sham animals, which was also observed in our study (Fig. 8C–F). Our findings indicate that G-CSF acts only on the injured sciatic nerve, and is an effective targeted therapy for treating neuropathic pain.

4.2.2 The mechanisms of antinociception by treating with G-CSF

We also show here that G-CSF increases the migration of opioid-containing PMN cells to sites of nerve injury and attenuates thermal hyperalgesia and mechanical allodynia (Fig. 8,11). Although the

precise underlying mechanisms remain unclear, G-CSF treatment induces peripheral analgesia probably via the secretion of opioid peptides from PMN cells. Secretion of opioids can also be stimulated by cold water swim stress or local injection of CRF and can subsequently activate the peripheral sensory neurons (Brack et al., 2004c). Therefore, G-CSF treatment may lead to an increase in the number of opioid-containing PMN cells, which can secrete opioids and attenuate neuropathic pain.

Moreover, the functional integrity of the immune system is essential for peripheral antinociception. In addition, endorphins help regulate the immune system and have neuroprotective properties (Kapitzke et al., 2005, Labuz et al., 2010).

An 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), in our results, we also found that μ-receptor protein was identified at the sciatic nerve in CCI-operated and sham-operated rats at day 1 (Fig. 13). Thus, G-CSF may act by increasing the secretion of endogenous opioids, which in turn act on the opioid receptors of injured nerves. Our study showed similar findings, because local application of NLXM could block the G-CSF-related analgesic effects (Fig. 14). Our results demonstrate that the effect of G-CSF on alleviating thermal hyperalgesia and mechanical allodynia is opioid/opioid receptor interaction-dependent; however, NLXM blocks the antinociception effects of G-CSF, which last for only 1 h, indicating that the alleviation of thermal hyperalgesia and mechanical allodynia by G-CSF is mediated through a continuous and complex mechanism.

In contrast, some studies have shown that G-CSF could not produce significant antinociception in inflammatory pain, cancer pain, or neuropathic pain in animal models (Schweizerhof et al., 2009, Liou et al., 2011). However, many aspects of the differences between those studies and ours require discussion. (1) The injection methods (subcutaneous [s.c.]

versus i.v.) and injection time and durations are different. In the study by Liou, it was expected that an increase in the number of subcutaneous injections may induce more inflammation in the animals. G-CSF has been demonstrated to have a therapeutic window (0–48 h) in this study, and it will crucially affect the long-term pain behaviors of animals with nerve injury. Thus, the G-CSF injection time-point at 72 h after CCI showed no significant effect on thermal hyperalgesia, but continued to produce inflammatory effects after that period (2) Cancer pain is a more and chronic process; therefore, peripheral tissue may become adapted to the stimuli and produce less CRF and fewer chemokines (ex. CXC family).

Thus, the analgesic effects of G-CSF may be less apparent without sufficient CRF and CXC chemokines.

Several peripheral endogenous antinociceptive mechanisms are involved in counteracting inflammatory hyperalgesia (Dale et al., 2005, Poisbeau et al., 2005, Mousa et al., 2007). Under inflammatory conditions, leukocytes secrete opioid peptides that bind to opioid receptors on peripheral sensory neurons and mediate antinociception (Cabot et al., 2001, Mousa et al., 2004, Rittner et al., 2006b, Rittner et al., 2008).

G-CSF is potentially important for the development of the immune and nervous systems, but its effects on neuropathic pain have not been fully

elucidated. We suggest that high doses of G-CSF via different routes might be able to deliver its neuroprotective effects in vivo. Moreover, G-CSF treatment can increase the number of leukocytes (Iwasaka et al., 2001) and enhance T-cell cytokine secretion (Brack et al., 2004d, Rutella, 2007). G-CSF also exerts some neuroprotective actions through the inhibition of apoptosis and inflammation as well as through the stimulation of neurogenesis (Solaroglu et al., 2007), which may also contribute toward alleviating neuropathic pain. Alternatively, G-CSF modulates the micro-environment in inflammatory sites, including cytokine expression profiles, resulting in enhanced cell survival, proliferation, and differentiation into cells of the neural lineage of bone marrow-derived stem cells that migrate into the lesion site (Tsai et al., 2008).

4.2.3 G-CSF regulates the pro-inflammatory cytokines in the DRG

Cytokines appear to induce acute-phase protein synthesis and are therefore a sensitive marker of tissue injury. Development of neuropathic pain is associated with multiple changes in gene expression in the DRG, which are modulated by inflammatory cytokines (Nilsson et al., 2005). In particular, IL-1, IL-6, and TNF-α play important roles in neuropathic pain in humans (Finley et al., 2008) by promoting the progression of secondary injury in the acute phase of nerve injury (Zhang et al., 2005, White et al., 2007). Moreover, IL-6 and TNF-α can activate microglial

cells in the DH (Marchand et al., 2005). G-CSF can suppress the in vitro expression of inflammatory cytokines and modulate their expression in experimental allergic encephalitis (Lazarini et al., 2003). In our study, IL-6 mRNA and TNF-α protein expression were suppressed in the G-CSF-treated group compared to the vehicle-treated group (Fig. 15A), which is consistent with the above findings (Fig. 8B). The DRG IL-6 mRNA and TNF-α protein levels increased immediately after the operation and then decreased slowly. Our results suggest that the relatively low inflammatory cytokine protein expression is related to less inflammation and pain.

4.2.4 G-CSF reduces the central neuroinflammation in the spinal DH

Sensitization of the spinal DH neurons leads to prolonged enhancement of pain behaviors that can be evoked by intense C-fiber stimulation, tissue inflammation, and peripheral nerve injury (Hathway et al., 2009). There is evidence that activated microglia OX-42 immunoreactivity is increased in the rat spinal cord following peripheral nerve injury in a time course that correlates with pain behaviors. Since

在文檔中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 (頁 49-69)