Figure 7 depicts the expression of HSP72 in sciatic nerve after CCI in different groups. It can be seen that the levels of HSP72 is significantly increased in sciatic nerve after 3 weeks exercise training program (Fig. 7A). The level of HSP72 increased to 3.15 ± 0.77 (P < 0.05, n=5) in CCITE rats after 3 weeks treadmill exercise training. However, the level of HSP72 increased insignificantly (1.92 ± 0.19, n=5) after 3 weeks swimming exercise training. After exercise training 39 days, the level of HSP72 significantly increased in CCTE group compared to CCI group. After exercise training 39 days, the level of HSP72 significantly increased in CCTE group compared to CCI group, about 3.9- fold (P <
0.05, n=4) as shown in Figure 7B. In addition, the level of HSP72 increased insignificantly (0.8- fold, n=4) after swimming exercise training 39 days.
3.5. Histopathological examination
Histological appearance was essentially normal for all sham operated groups, with a similar
16
distribution of small and large diameter nerve fibers, blood vessels and immune cells into one or more fascicles, each surrounded by a well-defined perineurium, and a regular proportion between myelin sheath thickness and fiber diameter as shown in Figure 8AE for comparison. Axonal
degenerations were evident in the sciatic nerve in CCI, CCISE and CCITE rats on D22 and D40, with a large amount of myelin, cell debris and immune cells as well as Wallerian degnereration of nerve fibers (Fig. 8). After exercise training 21 days and 39 days, the extent of degeneration significantly attenuated in CCTE (fig. 8CG) and CCISE (fig. 8DH) rats compared to CCI rats (fig. 8BF). Figure 8I shows the mean number of nuclei in a complete transverse section of sciatic nerve at different times (day 21 and day 40), expressed as a percentage of the mean number of nuclei in degenerated nerves.
17
4. Discussion
We found that swimming and treadmill exercises retarded peripheral neuropathic pain and protect nerve damage following chronic constriction injury of sciatic nerve in rats. Swimming and treadmill exercise decreased TNF-α and IL-1β expression and increased HSP72 expression in sciatic nerve after CCI-treatment.
In this study, rats with exercise (swimming or treadmill) showed body weight loss compared with rats without exercise. However, exercise did not affect the curves of body weight increases after CCI-treatment (Fig. 1AB). We thought that rats suffered from stress (e.g., exercise and CCI) and maintained similar curves of body weight increases.
Regardless of swimming or treadmill exercise, they retarded the decreasing curves of time courses of hyperalgesia and allodynia. CCI rat with swimming or treadmill exercise attenuated thermal hyperalgesia and mechanical allodynia significantly, when compared with CCI rats with exercise 21 days after CCI-treatment (Figs. 3 and 4). These results are agreed with previous studies, which reported swimming exercise (Kuphal et al. 2007) attenuated peripheral neuropathic pain in rats.
Treadmill and swimming exercise ameliorated spinal cord injury-induced allodynia and restored normal sensation after spinal cord contusion in rats (Hutchinson et al. 2004).
Neuropathic pain exhibit a series of anatomical, morphological and functional changes occur following damage to the peripheral nerve injury. Evidence has been presented that neuropathic pain consequent peripheral nerve injury is associated with local inflammation and overexpression of inflammation cytokines (Martucci et al. 2008). The present results are consistent with several
18
previous studies in partly. Previous studies have demonstrated that CCI induce activity in axons became hypersensitivity and enhanced transmission retrogradely to cell bodies in the dorsal root ganglia and spinal cord with subsequently released some mediators. For example, these mediators were able to activate the microglia cell via specific receptors and induce phosphorylation of p38 mitogen-activated protein kinase in spinal cord, where they may alter gene expression of the neurons (Tsuda et al. 2004; Song et al. 2005; Zhang et al. 2005; Burnstock 2006; Inoue 2006; Gu et al. 2008).
However, the hyperactive microglia result in the release of bioactive substances, including cytokines, prostaglandin E2 and excitatory amino acids (such as glutamate and aspartate) that alter the responses of dorsal horn cells and maintain the neuropathic pain states (Campbell and Meyer 2006; Inoue 2006).
Our results showed that treadmill or swimming exercise training could attenuate TNF-α and IL-1β expression 21 day after chronic constriction injury. This evidence may provide a reasonable explanation for our experimental results why exercise training could alleviate neuropathic pain following CCI in rats.
In agreement with our results, numerous studies demonstrate that exercise has beneficial effects on chronic disease, presents the neuroprotection, anti-inflammatory effect, and neuropathic pain resolution (Woods et al. 2006; Kuphal et al. 2007). Previous studies have demonstrated that
exercise-induced modulation of heat shock factor-1 (HSF-1, a HSPs transcription factor) aggregation, subsequently expression of HSP72 in multiple organs or neurons of rats (Hung et al. 2005; Chen et al.
2007; Noble et al. 2008; Hu et al. 2009). In addition, treatment with BRX-220 (co-inducer of HSPs) on the expression of HSP70 lead to slowly developing analgesic actions to allodynia and
19
enhancement of recovery processes on rat following L5 spinal nerve ligation (Kalmar et al. 2003).
Moreover, the increase in HSPs expression can decrease the production of the proinflammatory cytokines has been proved (Saleh et al. 2000). Our results showed that swimming or treadmill exercise training significantly promoted HSP72 expression and ameliorated the CCI-induced expression of proinflammatory cytokines (TNF-α and IL-1β) in sciatic nerve of rats, subsequently improved CCI-induced neuropathic pain. We suggest that the protective effect of HSP72 observed in this study is induced by the exercise mediated aggregation of HSF-1, consequently promoting HSP72 generation in the CCI rat. Although we do not provide direct evidence of the action mechanism of HSP72 attenuated proinflammatory cytokines expression in this study, accumulated evidences showed that HSPs can decrease the production of the proinflammatory cytokines (Saleh et al. 2000).
However, these observations on thermal hyperalgesia, mechanical allodynia, and HSP70 are, at present, merely co-incident.
Our neuropathological findings showed serious Wallerian degeneration, and axonal, and myelin damage after CCI. Previous studies indicates that Wallerian degeneration leads to release of
cytokines or other molecules from denervated Schwann cells, mast cells or other cell types that supports the concept that inputs from the peripheral nerve play ongoing critical role with regard to neuropathic pain (Lancelotta et al. 2003). In addition, axonal demyelination and degeneration were similar to those previous studies (Chen et al. 1992; Kato et al. 2002; Mazzer et al. 2008). Conversely, the level of degeneration was decreased by exercise training in the present study. Moreover,
regeneration provides reinnervation of these cells and is associated with decreasing hyperalgesia
20
(Lancelotta et al. 2003). In this study, exercise training may reduce Wallerian degeneration which leads to release of cytokines (TNF-α and IL-1β ) and increase regeneration which decreased hyperalgesia.
However, there is a limitation of this study. Treadmill and swimming exercise trainings in this study are forced, whereas they in humans are voluntary (Kuphal et al. 2007). Therefore, we must be careful when the findings in this study translate rodent therapeutic strategy to human cases.
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5. Conclusions
Our study demonstrated that swimming and treadmill exercise partially ameliorates thermal hyperalgesia, mechanical allodynia, TNF-α and IL-1β expression in sciatic nerve. Treadmill exercise, but not swimming exercise, increased HSP72 expression in sciatic nerve of CCI with exercise rats, compared with CCI without exercise rats. The behavioral improvement with daily exercise treatment may suggest a progressive analgesic action. These results of this study also indicated that treatment with exercise can be beneficial in Wallerian degeneration, axonal and myelin damage of sciatic nerve.
On the basis of our results we suggest that exercise might represent a potential therapeutic strategy for CCI-induced peripheral neuropathy.
Further studies are required to examine the possible mechanisms of activation, and to investigate the biological actions of HSP72 increased expressions responses to varying types of exercise training.
22
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Table 1 Graded Swimming Exercise Protocols
Day(s)
Exercise Period (min)
Rest Period
(min) Sessions
Total Exercise Duration (min)
-2~1 10 15 9 90
2 15 15 6 90
3 30 15 3 90
4 45 15 2 90
5 60,30 15 2 90
6 75,15 15 2 90
7-39 90 0 1 90
All swimming animals accommodate the water depth and temperature two days before surgery.
Chronic constriction injury and sham operation were performed on day 0. Behavioral assessments were measured on 1 day before and before surgery, after surgery day 1, 3, 7, 14, 21, 28, 35 and 39.
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Table 2 Graded Treadmill Exercise Protocols
The rats were run on a treadmill 5 days a week for 6 weeks. On the first week, all rats acclimatized the track and ran for 15 min at 1.2 km/hr, 0% slope for 3 days. The duration and intensity of the exercise were increased progressively. Chronic constriction injury and sham operation were
performed on day 0. Behavioral assessments were measured on 1 day before and before surgery, after surgery day 1, 3, 7, 14, 21, 28, 35 and 39.
Week(s) Day(s) Exercise Rate (km/hr) Total Exercise Duration (min)
1* day -2 to 4 1.2 15/ 30
2* day 5 to 11 1.8 30
3* day 12 to 18 1.8 60
4* day 19 to 25 1.8 60
5* day 26 to 32 1.8 60
6* day 33 to 39 1.8 60
30
B
Time after Surgery (days)-1 0 1 3 7 14 21 28 35 39
Fig. 1. Body weight change of treadmill (A) and swimming (B) exercised or non-exercised training on control, sham operated (SO) and neuropathic rats (CCI). Data represented as mean ± S.E.M of 8 to 10 rats per group. Symbols (*,**,***) indicate P < 0.05, P < 0.01, P < 0.001 when SO compare to CCI; (#.##,###) indicated P < 0.05, P < 0.01, P < 0.001 when SO compare to SOTE or SOSE; (+,++) indicated P < 0.05, P < 0.01 when CCI compare to CCISE. (one-way ANOVA followed by post hoc Tukey’s test) (SO: sham operation; SOTE: sham operation with treadmill exercise training; SOSE:
sham operation with swimming exercise training; CCI: chronic constriction injury; CCITE: chronic constriction injury with treadmill exercise training; CCISE: chronic constriction injury with
swimming exercise training)
31
A
-1 0 1 3 7 14 21 28 35 39
Paw Withdrawal Latency (sec)
0
B
Time after Surgery (days)-1 0 1 3 7 14 21 28 35 39
Fig. 2. Time courses of thermal hyperalgesia (A) and mechanical allodynia (B) in control, sham operated (SO) rats and sham operated exercise (SOTE or SOSE) rats. (see Fig. 1 abbreviations). The paw withdrawal latency (s) and pressure (g) to heat and mechanical stimulation were no significant differences among these groups compare to control, respectively. Data represented as mean ± S.E.M of 8 to 10 rats per group. (one-way ANOVA followed by post hoc Tukey’s test)
32
A
-1 0 1 3 7 14 21 28 35 39
Paw Withdrawal Latency (sec)
0
B
Time after Surgery (days)-1 0 1 3 7 14 21 28 35 39
Fig. 3. Time courses of thermal hyperalgesia (A) and mechanical allodynia (B) in CCI and CCITE rats. (see Fig. 1 abbreviations). Data represented as mean ± S.E.M of 6 to 8 rats per group. * P< 0.05,
** P< 0.01, as compared to CCI (Student’s t test)
33
A
-1 0 1 3 7 14 21 28 35 39
Paw Withdrawal Latency (sec)
0
B
Time after Surgery (days)-1 0 1 3 7 14 21 28 35 39
Fig. 4. Time courses of thermal hyperalgesia (A) and mechanical allodynia (B) in CCI and CCISE rats. (see Fig. 1 abbreviations). Data represented as mean ± S.E.M of 6 to 8 rats per group. * P< 0.05 as compared to CCI (Student’s t test)
34
Fig. 5. The level of TNF-α (A) and IL-1β (B) in sciatic nerve on D22 in different groups of rats: SO, CCI, CCITE, and CCISE. (see Fig. 1 abbreviations). The values represented mean ± S.E.M. of 5 rats per group. * P < 0.05, *** P < 0.001 as compared to SO; # P<0.05, ##P<0.01 as compared to CCI (one-way ANOVA followed by post hoc Tukey’s test)
A
B
35
Fig. 6. The level of TNF-α (A) and IL-1β (B) in sciatic nerve on D40 in different groups of rats: SO, CCI, CCITE, and CCISE. (see Fig. 1 abbreviations). The values represented mean ± S.E.M of 5 rats per group. * P<0.05,*** P<0.001 versus SO; # P<0.05, ### P<0.001 versus CCI (one-way ANOVA followed by post hoc Tukey’s test)
SO CCI CCITE CCISE
36
CCI CCITE CCISE
HSP72 (ratio to CCI group)
0
HSP72 (ratio to CCI group)
0
Fig. 7. The level of HSP72 in sciatic nerve on D22 (A) and D40 (B) in different groups of rats: CCI, CCITE and CCISE. (see Fig. 1 abbreviations). The values represented mean ± S.E.M. of 5 rats per group. * P<0.05 as compared to CCI. (one-way ANOVA followed by post hoc Tukey’s test)
72 kDa
37
D22 D40
A E
SO
B F
CCI
C G
CC ITE
D H
CCI S E
Fig. 8. The histopathological examination of the sciatic nerve on D22 and D40 in different groups of rats: SO (A,E), CCI (B,F), CCITE (C,G) and CCISE (D,F) groups. H&E stain 400X. (Scale bar = 200μm) Arrow ( ) indicates nuclei of immune cells; arrowhead ( ) indicates blood vessels.
38
CCI CCITE CCISE
% nuclei number (CCI/ SO nerve)
0 200 400 600 800 1000
D22 D40
*** ***
# # #
Fig. 9. Quantitative histopathology of Wallerian degnereration in the sciatic nerve sectioned transversely at 4μm. The values represented mean ± S.E.M. of 3 sections per group. (Non-parametric
Mann-Whitney U test) *** P < 0.001 as compared to CCI on D22; # # # P < 0.001 as compared to CCI on D40