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.
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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.
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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
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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.
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3.2 Part II
After unilateral CCI of the sciatic nerve, animals developedthermal 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 allodyniawere 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 allodyniacompared 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 wasinjected 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. DelayedG-CSF injections (24 and 48 h after CCI) resulted in a significantreversal of thermal hyperalgesia compared to the effect of only
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vehicle treatment (p < 0.05) (Fig. 9A, B). These findings are similar to those in rats with immediate G-CSF injections, which significantly attenuatedthermal hyperalgesia. However, delayedG-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
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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
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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 developedthermal 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.
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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).
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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).
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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
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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.
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