臺灣博碩士論文加值系統

背景及目的:經皮神經電刺激可增加糖尿病病患的冷及熱疼痛閾並減輕病患的疼痛指數，然而對於經皮神經電刺激是如何減輕糖尿病所引發的神經性疼痛目前仍未可知。不過先前的研究已指出高血糖會改變腫瘤壞死因子α(TNF-α)、腫瘤壞死因子受體1(TNFR1)、腫瘤壞死因子受體2(TNFR2)、半胱氨酸蛋白酶3(Caspase-3)、磷脂酰肌醇3-激酶(PI3k)、蛋白質激酶 B(Akt)在周邊神經中的濃度。本篇研究主要是去探討經皮神經電刺激對於糖尿病鼠疼痛行為的影響並去了解上述物質在背根神經節、脊髓、坐骨神經及周邊神經的濃度變化情況。方法:將大鼠隨機分在五個不同的組別，分別為控制組、糖尿病組、糖尿病用高頻電刺激治療組、糖尿病用低頻電刺激治療組及糖尿病用交替高低頻電刺激治療組，治療組治療時間皆為每天三十分鐘，每周五天，一共治療三周。兩側的後腳觸覺及熱覺疼痛閾都會進行測量以比較治療前後的差異。在引發糖尿病後的第二十一天會將大鼠犧牲，之後會將相對應於腰椎第四到六節的脊髓後角、背根神經節、坐骨神經及周邊神經(脛神經、腓總神經及腓腸神經)取下進行組織分析。腫瘤壞死因子α的表現量是用酶聯免疫吸附試驗進行分析，而腫瘤壞死因子受體1、腫瘤壞死因子受體2、半胱氨酸蛋白酶3、磷脂酰肌醇3-激酶、蛋白質激酶 B則用西方點墨法測量。結果:單次治療後觸覺刺激反應閾及熱刺激反應閾的最高點在半小時到兩小時之間，二十四小時後反應閾會些微下降。在三周的治療中，經皮神經電刺激能有效的增加觸覺反應閾，但只有高頻電刺激能有效的增加熱刺激反應閾。腫瘤壞死因子α經過三周的經皮神經電刺激治療後在脊髓後角和背根神經節有顯著性的下降，不過在坐骨神經及周邊神經經過高頻及低頻電刺激治療後都是上升的，只有交替高低頻組的腫瘤壞死因子α在坐骨神經有下降。腫瘤壞死因子受體1、腫瘤壞死因子受體2、半胱氨酸蛋白酶3、磷脂酰肌醇3-激酶、蛋白質激酶 B在脊髓後角的含量在糖尿病組及三個治療組中是有上升的。在背根神經節中，腫瘤壞死因子受體1、腫瘤壞死因子受體2及蛋白質激酶 B在糖尿病組及三個治療組中是有上升的。到了坐骨神經，在糖尿病組中就只剩下腫瘤壞死因子受體2和磷脂酰肌醇3-激酶是上升的，而在周邊神經中則沒有物質是上升的。在三個治療組中，壞死因子受體1(TNFR1)和腫瘤壞死因子受體2在坐骨神經中有上升，在周邊神經只有壞死因子受體1上升。結論:三種不同頻率的經皮神經電刺激皆可減輕糖尿病所引起的觸覺異常性疼痛，但在腫瘤壞死因子α及其下游蛋白質濃度中交替高低頻電刺激治療組及低頻電刺激治療組較高頻電刺激組的影響更為顯著。

Background and Purpose: Transcutaneous electrical nerve stimulation (TENS) has been used to improve heat and cold pain thresholds and decrease the pain scales in diabetic patients. However, it remains unknown whether TENS could attenuate diabetic neuropathy. Previous research has shown that hyperglycemia could change the levels of TNF-α, TNFR1, TNFR2, cleaved caspase-3, p-Akt and p-PI3k in the PN. Thus, this study investigates the effects of TENS in the pain-like behaviors of the diabetic rats and the expression of TNF-α, TNFR1, TNFR2, cleaved caspase-3, p-Akt and p-PI3k in the DRG, SCDH, SN and PN. Methods: Five experimental groups consist of control, STZ, STZ+ HF TENS, STZ+ LF TENS, and STZ+ AF TENS. TENS will be delivered to the HF, LF and AF groups for 30 minutes per day and 5 days per week for 3 weeks. The mechanical and thermal pain thresholds have been tested under the rats’ hind paws of each side. The rats were sacrificed on the 21st day since the diabetic induction. After the 30-minute post-test period of the last treatment, the L4-L6 SCDH, DRGs, SN and PN (including tibial nerve, peroneal nerve and sural nerve) are collected for protein analysis. TNF-α levels were detected by Enzyme-linked Immunosorbent Assay (ELISA) and TNFR1, TNFR2, cleaved caspase-3, p-Akt and p-PI3k expressions were analyzed by Western Blot. Results: The peaks of the mechanical withdrawal threshold and thermal withdrawal latency are shown lasting after 30 minutes until 2 hours of treatment, but they slightly decreased after 24 hours. TENS can effectively increase the mechanical withdrawal threshold for 3 weeks. However, HF TENS only increases the thermal withdrawal latency for 3 weeks. TNF-α level in the spinal cord dorsal horn and dorsal root ganglions were suppressed after the 3-week TENS treatment, but it increased in the sciatic nerve and the peripheral nerve in both high and LF groups. TNF-α level in the sciatic nerve only decreased in the AF group. Expression of TNFR1, TNFR2, cleaved caspase-3, p-Akt and p-PI3k in the spinal cord dorsal horn significantly up-regulated in the STZ and treatment groups. Expressions of TNFR1, TNFR2 and p-Akt in dorsal root ganglion up-regulated in the STZ and treatment groups as well. However, only the expressions of TNFR2 and p-PI3k in sciatic nerve increased in the STZ group. The expressions of TNFR1 and TNFR2 in sciatic nerve and TNFR1 in peripheral nerve both enhanced in the treatment groups. Conclusion: HF, LF and AF TENS can all lower mechanical allodynia in diabetes, but the decrease in TNF-α level and cell proliferation and the increase of apoptosis in LF and AF groups are more significant than HF group.

Abbreviations…………………………………………………………..I
Abstract………………………………………………………………...II
Chinese Abstract……………………………………………………….IV
誌謝………………………………………………………………...….VI
List of Figures…………………………………………………………VIII
List of Tables…………………………………………………………..XII
Introduction…………………………………………………………….1
Methods………………………………………………………………..5
Results…………………………………………………………………14
Discussion……………………………………………………………..19
Conclusion……………………………………………………………..23
References……………………………………………………………..24
Figures…………………………………………………………………30
Tables………………………………………………………………….149

1.Backonja, M., et al., Gabapentin for the symptomatic treatment of painful neuropathy in patients with diabetes mellitus: a randomized controlled trial. JAMA, 1998. 280(21): p. 1831-6.
2.Schmader, K.E., Epidemiology and impact on quality of life of postherpetic neuralgia and painful diabetic neuropathy. Clin J Pain, 2002. 18(6): p. 350-4.
3.Benbow, S.J., M.E. Wallymahmed, and I.A. MacFarlane, Diabetic peripheral neuropathy and quality of life. Qjm, 1998. 91(11): p. 733-7.
4.Morrow, T.J., Animal models of painful diabetic neuropathy: the STZ rat model. Curr Protoc Neurosci, 2004. Chapter 9: p. Unit 9.18.
5.Calcutt, N.A., J.D. Freshwater, and A.P. Mizisin, Prevention of sensory disorders in diabetic Sprague-Dawley rats by aldose reductase inhibition or treatment with ciliary neurotrophic factor. Diabetologia, 2004. 47(4): p. 718-24.
6.Kim, S.H., J.K. Kwon, and Y.B. Kwon, Pain modality and spinal glia expression by streptozotocin induced diabetic peripheral neuropathy in rats. Lab Anim Res, 2012. 28(2): p. 131-6.
7.McQuay, H.J., et al., Systematic review of outpatient services for chronic pain control. Health Technol Assess, 1997. 1(6): p. i-iv, 1-135.
8.Kilinc, M., et al., Effects of transcutaneous electrical nerve stimulation in patients with peripheral and central neuropathic pain. J Rehabil Med, 2014. 46(5): p. 454-60.
9.Forst, T., et al., Impact of low frequency transcutaneous electrical nerve stimulation on symptomatic diabetic neuropathy using the new Salutaris device. Diabetes Nutr Metab, 2004. 17(3): p. 163-8.
10.Kumar, D. and H.J. Marshall, Diabetic peripheral neuropathy: amelioration of pain with transcutaneous electrostimulation. Diabetes Care, 1997. 20(11): p. 1702-5.
11.Kumar, D., et al., Diabetic peripheral neuropathy. Effectiveness of electrotherapy and amitriptyline for symptomatic relief. Diabetes Care, 1998. 21(8): p. 1322-5.
12.Moharic, M. and H. Burger, Effect of transcutaneous electrical nerve stimulation on sensation thresholds in patients with painful diabetic neuropathy: an observational study. Int J Rehabil Res, 2010. 33(3): p. 211-7.
13.Somers, D.L. and F.R. Clemente, Contralateral high or a combination of high- and low-frequency transcutaneous electrical nerve stimulation reduces mechanical allodynia and alters dorsal horn neurotransmitter content in neuropathic rats. J Pain, 2009. 10(2): p. 221-9.
14.DeSantana, J.M., et al., Effectiveness of transcutaneous electrical nerve stimulation for treatment of hyperalgesia and pain. Curr Rheumatol Rep, 2008. 10(6): p. 492-9.
15.Melzack, R. and P.D. Wall, Pain mechanisms: a new theory. Science, 1965. 150(3699): p. 971-9.
16.Kumar, V.N. and J.B. Redford, Transcutaneous nerve stimulation in rheumatoid arthritis. Arch Phys Med Rehabil, 1982. 63(12): p. 595-6.
17.Garrison, D.W. and R.D. Foreman, Decreased activity of spontaneous and noxiously evoked dorsal horn cells during transcutaneous electrical nerve stimulation (TENS). Pain, 1994. 58(3): p. 309-15.
18.Hollman, J.E. and B.J. Morgan, Effect of transcutaneous electrical nerve stimulation on the pressor response to static handgrip exercise. Phys Ther, 1997. 77(1): p. 28-36.
19.Yarnitsky, D. and J.L. Ochoa, Studies of heat pain sensation in man: perception thresholds, rate of stimulus rise and reaction time. Pain, 1990. 40(1): p. 85-91.
20.Magerl, W., et al., Roles of capsaicin-insensitive nociceptors in cutaneous pain and secondary hyperalgesia. Brain, 2001. 124(Pt 9): p. 1754-64.
21.G, D.E.D., Pain relief with interferential therapy. Aust J Physiother, 1982. 28(3): p. 14-8.
22.Sluka, K.A., et al., Spinal blockade of opioid receptors prevents the analgesia produced by TENS in arthritic rats. J Pharmacol Exp Ther, 1999. 289(2): p. 840-6.
23.Matsuo, H., et al., Early transcutaneous electrical nerve stimulation reduces hyperalgesia and decreases activation of spinal glial cells in mice with neuropathic pain. Pain, 2014. 155(9): p. 1888-901.
24.Skundric, D.S. and R.P. Lisak, Role of neuropoietic cytokines in development and progression of diabetic polyneuropathy: from glucose metabolism to neurodegeneration. Exp Diabesity Res, 2003. 4(4): p. 303-12.
25.Purwata, T.E., High TNF-alpha plasma levels and macrophages iNOS and TNF-alpha expression as risk factors for painful diabetic neuropathy. J Pain Res, 2011. 4: p. 169-75.
26.Yan, J.E., et al., Streptozotocin-induced diabetic hyperalgesia in rats is associated with upregulation of Toll-like receptor 4 expression. Neurosci Lett, 2012. 526(1): p. 54-8.
27.Saleh, A., et al., Tumor necrosis factor-alpha elevates neurite outgrowth through an NF-kappaB-dependent pathway in cultured adult sensory neurons: Diminished expression in diabetes may contribute to sensory neuropathy. Brain Res, 2011. 1423: p. 87-95.
28.Li, Y., et al., Curcumin attenuates diabetic neuropathic pain by downregulating TNF-alpha in a rat model. Int J Med Sci, 2013. 10(4): p. 377-81.
29.Bazzoni, F. and B. Beutler, The tumor necrosis factor ligand and receptor families. N Engl J Med, 1996. 334(26): p. 1717-25.
30.Locksley, R.M., N. Killeen, and M.J. Lenardo, The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell, 2001. 104(4): p. 487-501.
31.Rauert, H., et al., Membrane tumor necrosis factor (TNF) induces p100 processing via TNF receptor-2 (TNFR2). J Biol Chem, 2010. 285(10): p. 7394-404.
32.Schmeichel, A.M., J.D. Schmelzer, and P.A. Low, Oxidative injury and apoptosis of dorsal root ganglion neurons in chronic experimental diabetic neuropathy. Diabetes, 2003. 52(1): p. 165-71.
33.Montgomery, S.L. and W.J. Bowers, Tumor necrosis factor-alpha and the roles it plays in homeostatic and degenerative processes within the central nervous system. J Neuroimmune Pharmacol, 2012. 7(1): p. 42-59.
34.Vivanco, I. and C.L. Sawyers, The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer, 2002. 2(7): p. 489-501.
35.Bellacosa, A., et al., Activation of AKT kinases in cancer: implications for therapeutic targeting. Adv Cancer Res, 2005. 94: p. 29-86.
36.Yang, Z.Z., et al., Physiological functions of protein kinase B/Akt. Biochem Soc Trans, 2004. 32(Pt 2): p. 350-4.
37.Downward, J., PI 3-kinase, Akt and cell survival. Semin Cell Dev Biol, 2004. 15(2): p. 177-82.
38.Sugimoto, K., et al., Early changes in insulin receptor signaling and pain sensation in streptozotocin-induced diabetic neuropathy in rats. J Pain, 2008. 9(3): p. 237-45.
39.Yang, C., et al., Isoflurane anesthesia aggravates cognitive impairment in streptozotocin-induced diabetic rats. Int J Clin Exp Med, 2014. 7(4): p. 903-10.
40.Suehiro, K., et al., Relationship between noradrenaline release in the locus coeruleus and antiallodynic efficacy of analgesics in rats with painful diabetic neuropathy. Life Sci, 2013. 92(23): p. 1138-44.
41.Otto, K.J., et al., Insulin implants prevent the temporal development of mechanical allodynia and opioid hyposensitivity for 24-wks in streptozotocin (STZ)-diabetic Wistar rats. Pain Med, 2011. 12(5): p. 782-93.
42.Zhao, W.C., et al., Curcumin ameliorated diabetic neuropathy partially by inhibition of NADPH oxidase mediating oxidative stress in the spinal cord. Neurosci Lett, 2014. 560: p. 81-5.
43.Nam, J.S., et al., Effects of nefopam on streptozotocin-induced diabetic neuropathic pain in rats. Korean J Pain, 2014. 27(4): p. 326-33.
44.Kinoshita, J., et al., Impaired noradrenaline homeostasis in rats with painful diabetic neuropathy as a target of duloxetine analgesia. Mol Pain, 2013. 9: p. 59.
45.Li, W., P. Wang, and H. Li, Upregulation of glutamatergic transmission in anterior cingulate cortex in the diabetic rats with neuropathic pain. Neurosci Lett, 2014. 568: p. 29-34.
46.Banafshe, H.R., et al., Effect of curcumin on diabetic peripheral neuropathic pain: possible involvement of opioid system. Eur J Pharmacol, 2014. 723: p. 202-6.
47.Hong, S., et al., The TRPV1 receptor is associated with preferential stress in large dorsal root ganglion neurons in early diabetic sensory neuropathy. J Neurochem, 2008. 105(4): p. 1212-22.
48.Huang, T.J., et al., Neurotrophin-3 prevents mitochondrial dysfunction in sensory neurons of streptozotocin-diabetic rats. Exp Neurol, 2005. 194(1): p. 279-83.
49.Huang, Y., et al., The role of TNF-alpha/NF-kappa B pathway on the up-regulation of voltage-gated sodium channel Nav1.7 in DRG neurons of rats with diabetic neuropathy. Neurochem Int, 2014. 75: p. 112-9.
50.Chen, Y.W., et al., Treadmill Training Combined with Insulin Suppresses Diabetic Nerve Pain and Cytokines in Rat Sciatic Nerve. Anesth Analg, 2015.
51.Chen, Y.W., et al., Physical exercise induces excess hsp72 expression and delays the development of hyperalgesia and allodynia in painful diabetic neuropathy rats. Anesth Analg, 2013. 116(2): p. 482-90.
52.Wang, D., et al., Assessment of diabetic peripheral neuropathy in streptozotocin-induced diabetic rats with magnetic resonance imaging. Eur Radiol, 2015. 25(2): p. 463-71.
53.Ingaramo, P.I., et al., Tumor necrosis factor alpha pathways develops liver apoptosis in type 1 diabetes mellitus. Mol Immunol, 2011. 48(12-13): p. 1397-407.
54.van Horssen, R., T.L. Ten Hagen, and A.M. Eggermont, TNF-alpha in cancer treatment: molecular insights, antitumor effects, and clinical utility. Oncologist, 2006. 11(4): p. 397-408.
55.Devin, A., et al., The distinct roles of TRAF2 and RIP in IKK activation by TNF-R1: TRAF2 recruits IKK to TNF-R1 while RIP mediates IKK activation. Immunity, 2000. 12(4): p. 419-29.
56.Liu, Z.G., et al., Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell, 1996. 87(3): p. 565-76.
57.Schwenzer, R., et al., The human tumor necrosis factor (TNF) receptor-associated factor 1 gene (TRAF1) is up-regulated by cytokines of the TNF ligand family and modulates TNF-induced activation of NF-kappaB and c-Jun N-terminal kinase. J Biol Chem, 1999. 274(27): p. 19368-74.
58.Wang, C.Y., et al., NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. Science, 1998. 281(5383): p. 1680-3.
59.Chu, Z.L., et al., Suppression of tumor necrosis factor-induced cell death by inhibitor of apoptosis c-IAP2 is under NF-kappaB control. Proc Natl Acad Sci U S A, 1997. 94(19): p. 10057-62.
60.Kreuz, S., et al., NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling. Mol Cell Biol, 2001. 21(12): p. 3964-73.
61.Lee, H.H., et al., NF-kappaB-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc Natl Acad Sci U S A, 1999. 96(16): p. 9136-41.
62.Fotin-Mleczek, M., et al., Apoptotic crosstalk of TNF receptors: TNF-R2-induces depletion of TRAF2 and IAP proteins and accelerates TNF-R1-dependent activation of caspase-8. J Cell Sci, 2002. 115(Pt 13): p. 2757-70.
63.Kamiya, H., W. Zhangm, and A.A. Sima, Apoptotic stress is counterbalanced by survival elements preventing programmed cell death of dorsal root ganglions in subacute type 1 diabetic BB/Wor rats. Diabetes, 2005. 54(11): p. 3288-95.
64.Stenberg, L., et al., Expression of activating transcription factor 3 (ATF 3) and caspase 3 in Schwann cells and axonal outgrowth after sciatic nerve repair in diabetic BB rats. Neurosci Lett, 2012. 515(1): p. 34-8.
65.Thorburn, A., Death receptor-induced cell killing. Cell Signal, 2004. 16(2): p. 139-44.
66.Schneider, P. and J. Tschopp, Apoptosis induced by death receptors. Pharm Acta Helv, 2000. 74(2-3): p. 281-6.
67.Ware, C.F., S. VanArsdale, and T.L. VanArsdale, Apoptosis mediated by the TNF-related cytokine and receptor families. J Cell Biochem, 1996. 60(1): p. 47-55.
68.Costa, G.N., et al., Contribution of TNF receptor 1 to retinal neural cell death induced by elevated glucose. Mol Cell Neurosci, 2012. 50(1): p. 113-23.
69.Hennessy, B.T., et al., Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov, 2005. 4(12): p. 988-1004.
70.Nam, S.Y., et al., Akt/PKB activation in gastric carcinomas correlates with clinicopathologic variables and prognosis. Apmis, 2003. 111(12): p. 1105-13.
71.Jiang, Y., et al., Diabetes induces changes in ILK, PINCH and components of related pathways in the spinal cord of rats. Brain Res, 2010. 1332: p. 100-9.
72.Yu, J., et al., Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. Mol Cell Biol, 1998. 18(3): p. 1379-87.
73.Rodriguez-Viciana, P., et al., Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature, 1994. 370(6490): p. 527-32.
74.Kodaki, T., et al., The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol, 1994. 4(9): p. 798-806.
75.Baptista, A.F., et al., High- and low-frequency transcutaneous electrical nerve stimulation delay sciatic nerve regeneration after crush lesion in the mouse. J Peripher Nerv Syst, 2008. 13(1): p. 71-80.