糖尿病是世界上常見的疾病之一，糖尿病患者會增加肢體截肢的風險和增加帶狀皰疹的嚴重度。截肢後疼痛和帶狀皰疹後疼痛都是神經病變痛，兩者都很難治療且常會影響生活品質。在這次實驗中，我們在糖尿鼠身上實行腰5脊神經結紮術，誘發神經病變痛來模擬以上的情況。熱休克蛋白70在脊神經受傷後會增加表現，有抗發炎和神經保護的作用。我們假設神經受傷後，增加熱休克蛋白70的表現跟疼痛行為的變化有關聯性。本研究希望了解當糖尿病鼠和非糖尿病鼠實行腰5脊神經結紮術時，熱休克蛋白70的表現和疼痛感覺的關聯性。
我們使用Sprague-Dawley成鼠來進行實驗，隨機分成以下四組：糖尿病接受腰5脊神經結紮組，非糖尿病接受腰5脊神經結紮組，糖尿病對照組，和非糖尿病對照組。對照組是指有進行同樣的手術但是沒有實行腰5脊神經結紮。給予腹膜內注射streptozotocin來誘發產生糖尿病鼠。腰5脊神經結紮術在給予streptozotocin十四天後實行。我們利用機械性自動刺激分析儀來測試老鼠的機械性疼痛閥值，運用西方墨點法及免疫組織化學染色法來分析熱休克蛋白70量的變化。
研究發現糖尿病鼠和非糖尿病鼠在接受腰5脊神經結紮術後都有明顯的機械性痛覺過敏，尤其是在糖尿病鼠。在腰5脊神經結紮術後第七天和第十四天，背根神經節的熱休克蛋白70在糖尿病接受腰5脊神經結紮組和非糖尿病接受腰5脊神經結紮組都有顯著的上升，但是在糖尿病接受腰5脊神經結紮這組，其熱休克蛋白70上升的量較少。此外，在糖尿病接受腰5脊神經結紮和非糖尿病接受腰5脊神經這兩組中，我們發現熱休克蛋白70主要呈現在背根神經節的無髓鞘C fiber和細小有髓鞘A-δ fiber的神經元中，且糖尿病接受腰5脊神經結紮這組，其熱休克蛋白70上升的量較少。
我們研究結果顯示在腰5脊神經結紮後，糖尿病接受腰5脊神經結紮這組，其熱休克蛋白70上升的量較非糖尿病接受腰5脊神經結紮組為少，特別是在背根神經節C fiber和A-δ fiber的神經元中，同時伴隨著更嚴重的機械性痛覺過敏。

Diabetes mellitus is a common disease worldwide. Diabetic patients have an increased risk of limb amputation and more severe herpes zoster infections. Post-amputation pain and post-herpetic neuralgia are neuropathic pain, and both are difficult to treat and often affect quality of life. In the present study, we used the L5 spinal nerve ligation (SNL) model to induce neuropathic pain in diabetic rats to mimic the above situation. Heat shock protein 70 (HSP70) expression increased after a spinal nerve injury, providing an anti-inflammatory effect as well as a neuroprotective function. We hypothesized that this increased HSP70 expression after a spinal nerve injury may be associated with neuropathic pain. The aim of this study was to determine the different reactions of HSP70 expression and pain sensation after inducing L5 SNL in diabetic and non-diabetic rats.
We randomly assigned adult male Sprague-Dawley (SD) rats into four groups: diabetic with L5 SNL, non-diabetic with L5 SNL, diabetic (sham), and non-diabetic (sham) groups. The sham groups underwent the same surgical procedure but without L5 SNL. Diabetic rats were prepared by intraperitoneal injection of streptozotocin. After 14 days of streptozotocin injection, SNL was performed. We used a dynamic plantar aesthesiometer to assess mechanical withdrawal threshold. The HSP70 protein contents were analyzed by Western blotting and immunohistochemistry.
We found that both diabetic and non-diabetic rats after L5 SNL showed significant mechanical hyperalgesia, which is more prominent in diabetic rats. After day 7 and day 14 of L5 SNL, the expression of HSP70 in dorsal root ganglions (DRGs) increased in both diabetic with SNL and non-diabetic with SNL groups, but the extent of increase was lower in the diabetic with SNL group. Additionally, within both diabetic with L5 SNL and non-diabetic with L5 SNL groups, HSP70 was predominantly present in unmyelinated C-fiber and thinly myelinated A-δ fiber neurons in DRGs, and the diabetic with L5 SNL group exhibited less HSP70 expression.
Our results indicated that after L5 SNL, the diabetic with L5 SNL group had less increased expression of HSP70, specifically in C-fiber and A-δ fiber neurons in DRGs combined with more mechanical hyperalgesia, as compared to the non-diabetic with L5 SNL group.

論文審定書…………………………………………………….… i
誌謝…………….…………………………………………………. ii
中文摘要…………………………………………………………… iii
English abstract……………………………………………………… v
Content………………………………………………………………. vii
Introduction…………………………………………………………… 1
Methods……………………………………………………………… 6
Animal preparation……………………………………………… 6
L5 SNL …………………………………………………………… 7
Behavior assessment of mechanical withdrawal threshold…… 8
Western blotting………………………………………………… 8
Immunohistochemistry………………………………………… 10
Statistical analysis……………………………………………… 11
Results……………………………………………………………… 12
Discussion…………………………………………………………… 18
Conclusion…………………………………………………………… 25
References…………………………………………………………… 26
Figures………………………………………………………………… 37
Figure 1…………………………………………………………… 37
Figure 2…………………………………………………………… 39
Figure 3……………………………………………………….…… 41
Figure 4……………………………………………………….……. 43
Figure 5…………………………………………………………….. 45
Figure 6…………………………………………………………….. 47
Figure 7…………………………………………………………….. 49
Figure 8…………………………………………………………….. 51
Figure 9…………………………………………………………….. 53
Figure 10………………………………………………………….. 55

1. Barrett AM, Lucero MA, Le T, Robinson RL, Dworkin RH, Chappell AS: Epidemiology, public health burden, and treatment of diabetic peripheral neuropathic pain: a review. Pain Medicine 2007, 8(s2):S50-S62.
2. Benbow S, Chan A, Bowsher D, MacFarlane I, Williams G: A Prospective Study of Painful Symptoms, Small‐fibre Function and Peripheral Vascular Disease in Chronic Painful Diabetic Neuropathy. Diabetic Medicine 1994, 11(1):17-21.
3. Dobretsov M, Romanovsky D, Stimers JR: Early diabetic neuropathy: triggers and mechanisms. World journal of gastroenterology: WJG 2007, 13(2):175-191.
4. Harati Y: Diabetes and the nervous system. Endocrinology &Metabolism Clinics of North America 1996, 25(2):325-359.
5. Ilnytska O, Lyzogubov VV, Stevens MJ, Drel VR, Mashtalir N, Pacher P, Yorek MA, Obrosova IG: Poly (ADP-ribose) polymerase inhibition alleviates experimental diabetic sensory neuropathy. Diabetes 2006, 55(6):1686-1694.
6. Fox A, Eastwood C, Gentry C, Manning D, Urban L: Critical evaluation of the streptozotocin model of painful diabetic neuropathy in the rat. Pain 1999, 81(3):307-316.
7. Kamei J, Ohhashi Y, Aoki T, Kasuya Y: Streptozotocin-induced diabetes in mice reduces the nociceptive threshold, as recognized after application of noxious mechanical stimuli but not of thermal stimuli. Pharmacology Biochemistry and Behavior 1991, 39(2):541-544.
8. Raz I, Hasdai D, Seltzer Z, Melmed RN: Effect of hyperglycemia on pain perception and on efficacy of morphine analgesia in rats. Diabetes 1988, 37(9):1253-1259.
9. Calcutt N, Freshwater J, Mizisin A: Prevention of sensory disorders in diabetic Sprague-Dawley rats by aldose reductase inhibition or treatment with ciliary neurotrophic factor. Diabetologia 2004, 47(4):718-724.
10. Sadosky A, McDermott AM, Brandenburg NA, Strauss M: A review of the epidemiology of painful diabetic peripheral neuropathy, postherpetic neuralgia, and less commonly studied neuropathic pain conditions. Pain Practice 2008, 8(1):45-56.
11. Asea A: Mechanisms of HSP72 release. Journal of Biosciences 2007, 32(3):579-584.
12. Zhang Y, Wang YH, Zhang XH, Ge HY, Arendt-Nielsen L, Shao JM, Yue SW: Proteomic analysis of differential proteins related to the neuropathic pain and neuroprotection in the dorsal root ganglion following its chronic compression in rats. Experimental Brain Research 2008, 189(2):199-209.
13. Latchman DS: Heat shock proteins and cardiac protection. Cardiovascular Research 2001, 51(4):637-646.
14. Giffard RG, Han RQ, Emery JF, Duan M, Pittet JF: Regulation of apoptotic and inflammatory cell signaling in cerebral ischemia-the complex roles of heat shock protein 70. Anesthesiology 2008, 109(2):339-348.
15. Zheng Z, Kim JY, Ma H, Lee JE, Yenari MA: Anti-inflammatory effects of the 70 kDa heat shock protein in experimental stroke. Journal of Cerebral Blood Flow & Metabolism 2007, 28(1):53-63.
16. Tavaria M, Gabriele T, Kola I, Anderson RL: A hitchhiker's guide to the human Hsp70 family. Cell Stress & Chaperones 1996, 1(1):23-28.
17. Turturici G, Sconzo G, Geraci F: Hsp70 and its molecular role in nervous system diseases. Biochemistry Research International 2011, 2011, Article ID 618127:1-18.
18. Kamiya H, Zhangm W, Sima AAF: 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):3288-3295.
19. Kamiya H, Zhang W, Sima AAF: Degeneration of the Golgi and neuronal loss in dorsal root ganglia in diabetic BioBreeding/Worcester rats. Diabetologia 2006, 49(11):2763-2774.
20. Atalay M, Oksala NKJ, Laaksonen DE, Khanna S, Nakao C, Lappalainen J, Roy S, Hänninen O, Sen CK: Exercise training modulates heat shock protein response in diabetic rats. Journal of Applied Physiology 2004, 97(2):605-611.
21. Najemnikova E, Rodgers CD, Locke M: Altered heat stress response following streptozotocin-induced diabetes. Cell Stress & Chaperones 2007, 12(4):342-352.
22. Swiecki C, Stojadinovic A, Anderson J, Zhao A, Dawson H, Shea-Donohue T: Effect of hyperglycemia and nitric oxide synthase inhibition on heat tolerance and induction of heat shock protein 72 kDa in vivo. The American Surgeon 2003, 69(7):587-592.
23. Barutta F, Pinach S, Giunti S, Vittone F, Forbes JM, Chiarle R, Arnstein M, Perin PC, Camussi G, Cooper ME: Heat shock protein expression in diabetic nephropathy. American Journal of Physiology-Renal Physiology 2008, 295(6):F1817-F1824.
24. Woolf CJ, Mannion RJ: Neuropathic pain: aetiology, symptoms, mechanisms, and management. The Lancet 1999, 353(9168):1959-1964.
25. Woolf CJ, Ma Q: Nociceptors--noxious stimulus detectors. Neuron 2007, 55(3):353-364.
26. Komori N, Takemori N, Kim HK, Singh A, Hwang SH, Foreman RD, Chung K, Chung JM, Matsumoto H: Proteomics study of neuropathic and nonneuropathic dorsal root ganglia: altered protein regulation following segmental spinal nerve ligation injury. Physiological Genomics 2007, 29(2):215-230.
27. Rajdev S, Hara K, Kokubo Y, Mestril R, Dillmann W, Weinstein PR, Sharp FR: Mice overexpressing rat heat shock protein 70 are protected against cerebral infarction. Annals of Neurology 2000, 47(6):782-791.
28. Kalmar B, Greensmith L, Malcangio M, McMahon S, Csermely P, Burnstock G: The effect of treatment with BRX-220, a co-inducer of heat shock proteins, on sensory fibers of the rat following peripheral nerve injury. Experimental Neurology 2003, 184(2):636-647.
29. Chen YW, Li YT, Chen YC, Li ZY, Hung CH: Exercise Training Attenuates Neuropathic Pain and Cytokine Expression After Chronic Constriction Injury of Rat Sciatic Nerve. Anesthesia & Analgesia 2012, 114(6):1330-1337.
30. Junger H, Sorkin LS: Nociceptive and inflammatory effects of subcutaneous TNFα. Pain 2000, 85(1):145-151.
31. Sommer C, Kress M: Recent findings on how proinflammatory cytokines cause pain: peripheral mechanisms in inflammatory and neuropathic hyperalgesia. Neuroscience Letters 2004, 361(1-3):184-187.
32. Ferreira S, Lorenzetti B, Bristow A, Poole S: Interleukin-1β as a potent hyperalgesic agent antagonized by a tripeptide analogue. Nature 1988, 334(6184):698-700.
33. Urban MJ, Li C, Yu C, Lu Y, Krise JM, McIntosh MP, Rajewski RA, Blagg BSJ, Dobrowsky RT: Inhibiting heat-shock protein 90 reverses sensory hypoalgesia in diabetic mice. ASN Neuro 2010, 2(4):189-199.
34. Yang ZC, Xia K, Wang L, Jia SJ, Li D, Zhang Z, Deng S, Zhang XH, Deng HW, Li YJ: Asymmetric dimethylarginine reduced erythrocyte deformability in streptozotocin-induced diabetic rats. Microvascular Research 2007, 73(2):131-136.
35. Zochodne DW, Verge VMK, Cheng C, Sun H, Johnston J: Does diabetes target ganglion neurones? Brain 2001, 124(11):2319-2334.
36. Ho Kim S, Mo Chung J: An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992, 50(3):355-363.
37. Hong S, Wiley JW: Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1. Journal of Biological Chemistry 2005, 280(1):618-627.
38. Girgis F, Wynn Parry C: Management of causalgia after peripheral nerve injury. Disability & Rehabilitation 1989, 11(1):15-20.
39. Patman R, Thompson JE, Persson AV: Management of post-traumatic pain syndromes: report of 113 cases. Annals of Surgery 1973, 177(6):780-787.
40. Donahue JG, Choo PW, Manson JAE, Platt R: The incidence of herpes zoster. Archives of Internal Medicine 1995, 155(15):1605-1609.
41. Opstelten W, Mauritz JW, De Wit NJ, Van Wijck AJM, Stalman WAB, Van Essen GA: Herpes zoster and postherpetic neuralgia: incidence and risk indicators using a general practice research database. Family Practice 2002, 19(5):471-475.
42. Heymann A, Chodick G, Karpati T, Kamer L, Kremer E, Green M, Kokia E, Shalev V: Diabetes as a risk factor for herpes zoster infection: results of a population-based study in Israel. Infection 2008, 36(3):226-230.
43. Rowbotham MC, Petersen KL: Zoster-associated pain and neural dysfunction. Pain 2001, 93(1):1-5.
44. Argoff CE, Katz N, Backonja M: Treatment of postherpetic neuralgia: a review of therapeutic options. Journal of Pain and Symptom Management 2004, 28(4):396-411.
45. Bild DE, Selby JV, Sinnock P, Browner WS, Braveman P, Showstack JA: Lower-extremity amputation in people with diabetes: epidemiology and prevention. Diabetes Care 1989, 12(1):24-31.
46. Reiber GE, Boyko EJ, Smith DG: Lower extremity foot ulcers and amputations in diabetes. Diabetes in America 1995, 2:409-428.
47. Nikolajsen L, Jensen TS: Phantom limb pain. British Journal of Anaesthesia 2001, 87(1):107-116.
48. Pucher I, Kickinger W, Frischenschlager O: Coping with amputation and phantom limb pain. Journal of Psychosomatic Research 1999, 46(4):379-383.
49. Davidson J: A survey of the satisfaction of upper limb amputees with their prostheses, their lifestyles, and their abilities. Journal of Hand Therapy 2002, 15(1):62-70.
50. Park HS, Lee JS, Huh SH, Seo JS, Choi EJ: Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase. Science's STKE 2001, 20(3):446-456.
51. Chung J, Nguyen AK, Henstridge DC, Holmes AG, Chan M, Mesa JL, Lancaster GI, Southgate RJ, Bruce CR, Duffy SJ: HSP72 protects against obesity-induced insulin resistance. Proceedings of the National Academy of Sciences 2008, 105(5):1739-1744.
52. Daviau A, Proulx R, Robitaille K, Di Fruscio M, Tanguay RM, Landry J, Patterson C, Durocher Y, Blouin R: Down-regulation of the mixed-lineage dual leucine zipper-bearing kinase by heat shock protein 70 and its co-chaperone CHIP. Journal of Biological Chemistry 2006, 281(42):31467-31477.
53. Gao YJ, Ji RR: Activation of JNK pathway in persistent pain. Neuroscience Letters 2008, 437(3):180-183.
54. Li N, Wang M, Oberley TD, Sempf JM, Nel AE: Comparison of the pro-oxidative and proinflammatory effects of organic diesel exhaust particle chemicals in bronchial epithelial cells and macrophages. The Journal of Immunology 2002, 169(8):4531-4541.
55. Doya H, Ohtori S, Fujitani M, Saito T, Hata K, Ino H, Takahashi K, Moriya H, Yamashita T: c-Jun N-terminal kinase activation in dorsal root ganglion contributes to pain hypersensitivity. Biochemical and Biophysical Research Communications 2005, 335(1):132-138.
56. Zhuang ZY, Wen YR, Zhang DR, Borsello T, Bonny C, Strichartz GR, Decosterd I, Ji RR: A peptide c-Jun N-terminal kinase (JNK) inhibitor blocks mechanical allodynia after spinal nerve ligation: respective roles of JNK activation in primary sensory neurons and spinal astrocytes for neuropathic pain development and maintenance. The Journal of Neuroscience 2006, 26(13):3551-3560.
57. Obata K, Yamanaka H, Kobayashi K, Dai Y, Mizushima T, Katsura H, Fukuoka T, Tokunaga A, Noguchi K: Role of mitogen-activated protein kinase activation in injured and intact primary afferent neurons for mechanical and heat hypersensitivity after spinal nerve ligation. The Journal of Neuroscience 2004, 24(45):10211-10222.
58. Chen H, Wu Y, Zhang Y, Jin L, Luo L, Xue B, Lu C, Zhang X, Yin Z: Hsp70 inhibits lipopolysaccharide-induced NF-κB activation by interacting with TRAF6 and inhibiting its ubiquitination. FEBS Letters 2006, 580(13):3145-3152.
59. Ran R, Lu A, Zhang L, Tang Y, Zhu H, Xu H, Feng Y, Han C, Zhou G, Rigby AC: Hsp70 promotes TNF-mediated apoptosis by binding IKKγ and impairing NF-κB survival signaling. Genes & Development 2004, 18(12):1466-1481.
60. Epstein FH, Barnes PJ, Karin M: Nuclear factor-κB—a pivotal transcription factor in chronic inflammatory diseases. New England Journal of Medicine 1997, 336(15):1066-1071.
61. Sun T, Song WG, Fu ZJ, Liu ZH, Liu YM, Yao SL: Alleviation of neuropathic pain by intrathecal injection of antisense oligonucleotides to p65 subunit of NF-κB. British Journal of Anaesthesia 2006, 97(4):553-558.
62. Ouyang YB, Xu LJ, Sun YJ, Giffard RG: Overexpression of inducible heat shock protein 70 and its mutants in astrocytes is associated with maintenance of mitochondrial physiology during glucose deprivation stress. Cell stress & Chaperones 2006, 11(2):180-186.
63. Feinstein DL, Galea E, Aquino DA, Li GC, Xu H, Reis DJ: Heat shock protein 70 suppresses astroglial-inducible nitric-oxide synthase expression by decreasing NFκB activation. Journal of Biological Chemistry 1996, 271(30):17724-17732.
64. Griendling KK, Ushio-Fukai M: Reactive oxygen species as mediators of angiotensin II signaling. Regulatory Peptides 2000, 91(1):21-27.
65. Kim HK, Park SK, Zhou JL, Taglialatela G, Chung K, Coggeshall RE, Chung JM: Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain. Pain 2004, 111(1):116-124.
66. Tal M: A novel antioxidant alleviates heat hyperalgesia in rats with an experimental painful peripheral neuropathy. Neuroreport 1996, 7(8):1382-1384.