| 摘要: | 研究背景及動機
周邊神經損傷引發的神經發炎反應及神經病理性疼痛，不但會造成病人生理和心理上的不適，也會間接的影響生活品質。低能雷射治療(low level laser therapy, LLLT)常被使用於減低疼痛、發炎、幫助傷口癒合和加速組織再生，但是對於有關探討周邊神經損傷的影響及分子生物機轉的文獻甚少。另外，近年來有文獻探討關於低氧因子(hypoxia inducible factor-1α, HIF-1α)在參與發炎的過程中也扮演了重要的角色，但在針對在周邊神經損傷所造成的發炎調控機轉，則尚未明確。所以研究利用慢性坐骨神經損傷緊縮性損傷的動物模式(chronic constriction injury, CCI)探討以低能雷射治療，對於周邊神經損傷的神經病理性疼痛及功能性恢復的影響。
研究目的
本研究的目的是為了探討LLLT是否在周邊神經傷害後，可以促進神經功能之恢復，以及LLLT在周邊神經病變上，是否可以藉由降低低氧誘發因子(hypoxia-inducible factor-1 alpha, HIF-1α)、促發炎因子(tumor necrosis factorα,TNF-α和interleukin-1β,IL-1β)、一氧化氮合成酶(inducible nitric oxide syntheses, iNOS)、還氧合酶(cyclooxygenase enzymes-2, COX-2)、腫瘤抑制蛋白(tumor suppressor protein p53, p53)及巨噬細胞的過度堆積，達到降低發炎反應及神經痛，並可促進生長因子血管內皮生長因子(vascular endothelial growth factor, VEGF),神經生長因子(nerve growth factor, NGF)和腦源性神經生長因子(brain-derived neurotrophic factor, BDNF) 和S100之表現，來促進CCI動物之功能性恢復。另外，亦加以探討LLLT的分子生物機制，是否有劑量依賴性(dose-dependent)的效果。
材料與方法
將120隻SD大鼠依據CCI手術、假性CCI手術(sham-operated CCI, sCCI)、LLLT介入及假性LLLT (sham-operated LLLT)隨機分組，並進一步依治療時間的安排再作次分組(subgroup)。因此，本實驗的分組(共12組)如下：(1) CCI手術與LLLT組 (CL group)：本組再分成CCI + LLLT治療一週(n=10)、兩週(n=10)及三週 (n=10); (2) CCI手術與sLLLT組 (CsL group)：本組再分成 CCI +sLLLT治療一週(n=10)、兩週(n=10)及三週 (n=10); (3) sCCI手術與LLLT組 (sCL group)：本組再分成sCCI + LLLT治療一週(n=10)、兩週(n=10)及三週 (n=10);及(4) sCCI手術與sLLLT組 (sCsL group)：本組再分成sCCI + sLLLT治療一週(n=10)、兩週(n=10)及三週 (n=10)。以連續性660-nm Ga-Al-As 低能雷射，輸出能力(output power) 為30 mW，在手術處的坐骨神經正上方，照射60秒，總計能量密度(energy densitie)為9J/cm2，連續照射一週、兩週和三週。我們會利用疼痛行為測試、坐骨神經功能指數 (sciatic function index, SFI)、脛神經功能指數(tibial function index, TFI)、腓神經功能指數(peroneal function index, PFI)及坐骨神經靜態指標 (sciatic static index, SSI )、組織切片染色型態分析(histopathological examination)、酵素聯結免疫吸附測試 (ELISA)和西方點墨法(Western blot)，來評估LLLT的治療效果。
結果
本實驗結果顯示，在CCI神經損傷後，LLLT顯著提升機械性疼痛閾值、坐骨神經功能指數SFI、TFI、PFI、SSI (CL vs. CsL: P<0.05)。LLLT亦可以顯著降低HIF-1α、TNF-α、IL-1β、iNOS、COX-2、p53及巨噬細胞的過度堆積 (CL vs. CsL: P<0.05)，並顯著促進生長因子VEGF, NGF, 和BDNF的表現量及促進神經髓鞘生成(CL vs. CsL: P<0.05)。且LLLT對於這些發炎因子及生長因子的影響而言，並無劑量依賴性（dose-dependent）的效果 (P>0.05)。
結論
在CCI神經損傷後，LLLT可以降低HIF-1α、TNF-α、IL-1β、iNOS、COX-2、p53及巨噬細胞的過度堆積，來達到減低神經性病理疼痛效果；並可促進生長因子VEGF, NGF, 和BDNF的表現量及促進神經髓鞘生成，來達到功能性恢復的效果。
Background
Peripheral nerve injury may induce nerve inflammation and neuropathic pain,and these phenomena not only cause physical and psychological discomfort of patients, but may also indirectly affect their quality of life. Low-energy laser treatment (low level laser therapy, LLLT) is often used to reduce pain, inflammation, wound healing and to help accelerate tissue regeneration, but the literatures and studies concerned about the therapeutic effects on the peripheral nerve injury and the molecular biological mechanism of LLLT were very few. In addition, in recent years, many researches explored that hypoxia inducible factor-1α (HIF-1α) involves in the process of inflammation, but its role and mechanism in regulation of peripheral nerve injury-induced inflammation was still not clear. Therefore, the animal model of sciatic chronic constriction injury (CCI) was used in this study for investigating the effects of LLLT on neuropathic pain and functional recovery.
Purpose
The purposes of this study were to assess the suppressive effects of LLLT on nerve function, accumulation of hypoxia-inducible factor-1 alpha (HIF-1α), proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factorα (TNF-α) , inducible nitric oxide syntheses (iNOS), cyclooxygenase enzymes-2 (COX-2), tumor suppressor protein p53 (p53) and macrophages for controlling neuropathic pain after peripheral injury. Moreover, the effects of LLLT on activation of vascular endothelial growth factor (VEGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and repair of injured myelin sheath for promoting functional recovery were also assessed in CCI model.
Methods
Totally 120 rats were divided randomly into four groups based on the surgical procedure of CCI (operated VS sham-operated) and the treatment (LLLT VS sham-operated LLLT).Each group was further divided into three subgroups according to the duration of LLLT treatment. The experimental groups in this study included (1) CCI + LLLT (CL group) included the 1-week (n=10), 2-week (n=10) and 3-week (n=10) of LLLT; (2) CCI+ sLLLT (CsL group) included the 1-week (n=10), 2-week (n=10) and 3-week (n=10) of sLLLT; (3) sCCI + LLLT (sCL group) included the 1-week (n=10), 2-week (n=10) and 3-week (n=10) of LLLT;(4) sCCI + sLLLT(sCsL group)：included the 1-week (n=10), 2-week (n=10) and 3-week (n=10) of sLLLT. Seven days after surgery, animals started using the continuous 660-nm Ga-Al-As diode laser, and the output power of the laser irradiation was 30 mW per session with irradiation time for 60 sec/ per spot. The energy densities were 9 J/cm2. Effects of LLLT in CCI animals were determined by the measurements of mechanical pain threshold, sciatic function index, (SFI), tibial function index (TFI), peroneal function index (PFI), sciatic static index (SSI ), histopathological and immunohistochemical examination, ELISA and Western blot analysis.
Histopathological and immunological assessements included accumulation of hypoxia-inducible factor-1 alpha (HIF-1α), proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factorα (TNF-α), inducible nitric oxide syntheses (iNOS), cyclooxygenase enzymes-2 (COX-2), tumor suppressor protein p53 (p53) and macrophages for controlling neuropathic pain.
Results
Our results demonstrated that LLLT significantly improved mechanical pain threshold, sciatic functional index SFI, TFI, PFI, SSI (CL vs. CsL: P <0.05) after CCI. LLLT can also significantly reduce overexpressions of HIF-1α, TNF-α, IL-1β, iNOS, COX-2, p53, and the excessive macrophage accumulation (CL vs. CsL: P <0.05), and significantly promote the amounts of VEGF, NGF, and BDNF and myelin sheath (CL vs. CsL: P <0.05). But there was no significant differences among 1-, 2- and 3-week of LLLT on the expression of these protein factors (P>0.05).
Conclusions
After CCI nerve injury, LLLT can reduce neuropathic pain by suppressing the HIF-1α, TNF-α, IL-1β, iNOS, COX-2, p53 accumulation and macrophages infiltration and promote functional recovery by increasing the amounts of VEGF, NGF, BDNF and myelin sheath. |
