成熟哺乳類動物的中樞神經系統在受到損傷後將失去再生復原的能力，雖然過去的研究已證實電刺激可以增進神經的再生，然而電刺激如何促進中樞神經系統再生的機制仍待更進一步的研究。本實驗將探討不同電刺激波形對金魚視網膜節細胞神經纖維再生的影響，並探究電刺激對神經細胞所產生的作用及其可能的機制。在培養視網膜組織一天後，發現施加一小時間歇性脈衝電波比連續方波或連續脈衝電波更能促進視網膜神經纖維的再生。實驗也證實電刺激是藉由啟動高電壓敏感性鈣離子通道而引發鈣離子流入細胞以促進神經再生。若是抑制PKC的活性，電刺激便無法產生作用，顯示電刺激是藉由影響PKC的反應來促進視網膜神經纖維的再生。另外，雖然電刺激和IGF-I同時施加能增加神經纖維的再生長度，但電刺激無法單獨使IGF-I受器被抑制的神經恢復再生，因此顯示電刺激是藉由引發和IGF-I相同的路徑以促進神經再生。總結來說，本篇研究結果證實間歇性脈衝電波對金魚視網膜節細胞神經纖維再生的助益最大，並且指出電刺激是經由高電壓性敏感鈣離子通道引發鈣離子流入細胞，進而引發鈣離子下游PKC和IGF-I的反應以促進神經纖維的再生。

Central nervous system (CNS) in adult mammals loses the ability to regenerate after injury. Although electrical stimulation (ES) has been shown to promote neural regeneration, the underlying mechanisms of how ES enhances CNS regeneration remain elusive. The present study was aimed to investigate the effect of ES waveforms on neurite outgrowth of goldfish retinal explants and to elucidate possible cellular mechanisms responsible for the observed ES effects. When the retinal explants were stimulated for one hour with intermittent pulses at Day 1, the regenerated neurite length was significantly increased compared to those stimulated with continuous square waves or continuous pulses on the same day. We also demonstrated that ES could induce calcium influx through voltage gated calcium channels, and the enhancement of neurite outgrowth after ES application was reduced by protein kinase C (PKC) inhibitor. Moreover, we found that Insulin-like growth factor I (IGF-I) did not further facilitate the ES effect, and ES alone did not rescue neurite outgrowth reduced by inactivating IGF-I receptors. These results suggest that intermittent pulse ES promotes neurite regeneration most effectively in goldfish retinal explants, and ES enhances neurite outgrowth by activating PKC and IGF-I via calcium influx through voltage gated calcium channels.

Abstract i
摘要 ii
1. Introduction 1
1.1 Axon regeneration in the mammalian central nervous system 1
1.2 The effects of electrical stimulation on nervous system regeneration 2
1.3 Molecular mechanisms of electrical stimulation in regenerating neurons 5
1.4 Axon regeneration in the goldfish visual system 7
1.5 Goals and approaches 8
2. Materials and Methods 9
2.1 Animals and optic nerve crush 9
2.2 Culture of retinal explants 9
2.3 Electrical stimulation 10
2.4 Image acqusition and data analysis 11
3. Results 12
3.1 Neurite outgrowth in goldfish retinal explants was normal in ITO conductive devices 12
3.2 Electrical stimulation significantly enhanced neurite outgrowth in goldfish retinal explants 12
3.3 The strength and repetitiveness of electrical stimulation increased and sustained neurite outgrowth in goldfish retinal explants, respectively 13
3.4 Electrical stimulation with intermittent pulses further promoted neurite outgrowth in goldfish retinal explants 13
3.5 Electrical stimulation induced calcium influx through voltage gated calcium channels to promote neurite outgrowth in goldfish retinal explants 14
3.6 Protein kinase C inhibitor reduced neurite outgrowth promoted by electrical stimulation in goldfish retinal explants 15
3.7 Insulin-like growth factor I did not further enhance neurite outgrowth promoted by electrical stimulation in goldfish retinal explants 15
4. Discussion 17
4.1 The waveform of electrical stimulation is a key factor in enhancing neurite outgrowth in goldfish retinal explants 17
4.2 The enhancement of neurite outgrowth by electrical stimulation is likely resulting from calcium influx through voltage gated calcium channels and activation of Protein kinase C and Insulin-like growth factor I 20
5. References 22
6. Figures 28

Al-Majed AA, Brushart TM, Gordon T (2000a) Electrical stimulation accelerates and increases expression of BDNF and trkB mRNA in regenerating rat femoral motoneurons. Eur J Neurosci 12:4381-4390.
Al-Majed AA, Tam SL, Gordon T (2004) Electrical stimulation accelerates and enhances expression of regeneration-associated genes in regenerating rat femoral motoneurons. Cell Mol Neurobiol 24:379-402.
Al-Majed AA, Neumann CM, Brushart TM, Gordon T (2000b) Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J Neurosci 20:2602-2608.
Bindokas VP, Ishida AT (1996) Conotoxin-sensitive and conotoxin-resistant Ca2+ currents in fish retinal ganglion cells. J Neurobiol 29:429-444.
Bito H (1998) The role of calcium in activity-dependent neuronal gene regulation. Cell Calcium 23:143-150.
Bito H, Deisseroth K, Tsien RW (1996) CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87:1203-1214.
Boucher SE, Hitchcock PF (1998) Insulin-related growth factors stimulate proliferation of retinal progenitors in the goldfish. J Comp Neurol 394:386-394.
Cai D, Shen Y, De Bellard M, Tang S, Filbin MT (1999) Prior exposure to neurotrophins blocks inhibition of axonal regeneration by MAG and myelin via a cAMP-dependent mechanism. Neuron 22:89-101.
Caroni P, Schwab ME (1988a) Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter. Neuron 1:85-96.
Caroni P, Schwab ME (1988b) Two membrane protein fractions from rat central myelin with inhibitory properties for neurite growth and fibroblast spreading. J Cell Biol 106:1281-1288.
Ciccolini F, Collins TJ, Sudhoelter J, Lipp P, Berridge MJ, Bootman MD (2003) Local and global spontaneous calcium events regulate neurite outgrowth and onset of GABAergic phenotype during neural precursor differentiation. J Neurosci 23:103-111.
Connor JA (1986) Digital imaging of free calcium changes and of spatial gradients in growing processes in single, mammalian central nervous system cells. Proc Natl Acad Sci U S A 83:6179-6183.
David S, Aguayo AJ (1981) Axonal elongation into peripheral nervous system "bridges" after central nervous system injury in adult rats. Science 214:931-933.
De Koninck P, Schulman H (1998) Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations. Science 279:227-230.
Dergham P, Ellezam B, Essagian C, Avedissian H, Lubell WD, McKerracher L (2002) Rho signaling pathway targeted to promote spinal cord repair. J Neurosci 22:6570-6577.
Dolmetsch RE, Xu K, Lewis RS (1998) Calcium oscillations increase the efficiency and specificity of gene expression. Nature 392:933-936.
Enes J, Langwieser N, Ruschel J, Carballosa-Gonzalez MM, Klug A, Traut MH, Ylera B, Tahirovic S, Hofmann F, Stein V, Moosmang S, Hentall ID, Bradke F (2010) Electrical activity suppresses axon growth through Ca(v)1.2 channels in adult primary sensory neurons. Curr Biol 20:1154-1164.
Fields RD, Neale EA, Nelson PG (1990) Effects of patterned electrical activity on neurite outgrowth from mouse sensory neurons. J Neurosci 10:2950-2964.
Fields RD, Eshete F, Stevens B, Itoh K (1997) Action potential-dependent regulation of gene expression: temporal specificity in Ca2+, cAMP-responsive element binding proteins, and mitogen-activated protein kinase signaling. J Neurosci 17:7252-7266.
Filbin MT (2003) Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nat Rev Neurosci 4:703-713.
Fujikado T, Morimoto T, Matsushita K, Shimojo H, Okawa Y, Tano Y (2006) Effect of transcorneal electrical stimulation in patients with nonarteritic ischemic optic neuropathy or traumatic optic neuropathy. Jpn J Ophthalmol 50:266-273.
Geremia NM, Gordon T, Brushart TM, Al-Majed AA, Verge VM (2007) Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression. Exp Neurol 205:347-359.
Goldberg JL, Espinosa JS, Xu Y, Davidson N, Kovacs GT, Barres BA (2002) Retinal ganglion cells do not extend axons by default: promotion by neurotrophic signaling and electrical activity. Neuron 33:689-702.
Gomez TM, Spitzer NC (1999) In vivo regulation of axon extension and pathfinding by growth-cone calcium transients. Nature 397:350-355.
Gomez TM, Zheng JQ (2006) The molecular basis for calcium-dependent axon pathfinding. Nat Rev Neurosci 7:115-125.
Gu X, Spitzer NC (1995) Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients. Nature 375:784-787.
Hanson MG, Landmesser LT (2004) Normal patterns of spontaneous activity are required for correct motor axon guidance and the expression of specific guidance molecules. Neuron 43:687-701.
Heacock AM, Agranoff BW (1997) Protein kinase inhibitors block neurite outgrowth from explants of goldfish retina. Neurochem Res 22:1179-1185.
Henley JR, Huang KH, Wang D, Poo MM (2004) Calcium mediates bidirectional growth cone turning induced by myelin-associated glycoprotein. Neuron 44:909-916.
Homma K, Koriyama Y, Mawatari K, Higuchi Y, Kosaka J, Kato S (2007) Early downregulation of IGF-I decides the fate of rat retinal ganglion cells after optic nerve injury. Neurochem Int 50:741-748.
Hong K, Nishiyama M, Henley J, Tessier-Lavigne M, Poo M (2000) Calcium signalling in the guidance of nerve growth by netrin-1. Nature 403:93-98.
Hopkins JM, Ford-Holevinski TS, McCoy JP, Agranoff BW (1985) Laminin and optic nerve regeneration in the goldfish. J Neurosci 5:3030-3038.
Kaneda M, Nagashima M, Nunome T, Muramatsu T, Yamada Y, Kubo M, Muramoto K, Matsukawa T, Koriyama Y, Sugitani K, Vachkov IH, Mawatari K, Kato S (2008) Changes of phospho-growth-associated protein 43 (phospho-GAP43) in the zebrafish retina after optic nerve injury: a long-term observation. Neurosci Res 61:281-288.
Kim JE, Li S, GrandPre T, Qiu D, Strittmatter SM (2003) Axon regeneration in young adult mice lacking Nogo-A/B. Neuron 38:187-199.
Kimura K, Yanagida Y, Haruyama T, Kobatake E, Aizawa M (1998) Gene expression in the electrically stimulated differentiation of PC12 cells. J Biotechnol 63:55-65.
Koriyama Y, Homma K, Sugitani K, Higuchi Y, Matsukawa T, Murayama D, Kato S (2007) Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration. Neurochem Int 50:749-756.
Li W, Llopis J, Whitney M, Zlokarnik G, Tsien RY (1998) Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature 392:936-941.
Lohmann C, Finski A, Bonhoeffer T (2005) Local calcium transients regulate the spontaneous motility of dendritic filopodia. Nat Neurosci 8:305-312.
Matsukawa T, Arai K, Koriyama Y, Liu Z, Kato S (2004a) Axonal regeneration of fish optic nerve after injury. Biol Pharm Bull 27:445-451.
Matsukawa T, Sugitani K, Mawatari K, Koriyama Y, Liu Z, Tanaka M, Kato S (2004b) Role of purpurin as a retinol-binding protein in goldfish retina during the early stage of optic nerve regeneration: its priming action on neurite outgrowth. J Neurosci 24:8346-8353.
McCaig CD (1987) Spinal neurite reabsorption and regrowth in vitro depend on the polarity of an applied electric field. Development 100:31-41.
Meyer RL (1980) Mapping the normal and regenerating retinotectal projection of goldfish with autoradiographic methods. J Comp Neurol 189:273-289.
Ming G, Henley J, Tessier-Lavigne M, Song H, Poo M (2001) Electrical activity modulates growth cone guidance by diffusible factors. Neuron 29:441-452.
Miyake K, Yoshida M, Inoue Y, Hata Y (2007) Neuroprotective effect of transcorneal electrical stimulation on the acute phase of optic nerve injury. Invest Ophthalmol Vis Sci 48:2356-2361.
Morimoto T, Miyoshi T, Sawai H, Fujikado T (2010) Optimal parameters of transcorneal electrical stimulation (TES) to be neuroprotective of axotomized RGCs in adult rats. Exp Eye Res 90:285-291.
Morimoto T, Miyoshi T, Matsuda S, Tano Y, Fujikado T, Fukuda Y (2005) Transcorneal electrical stimulation rescues axotomized retinal ganglion cells by activating endogenous retinal IGF-1 system. Invest Ophthalmol Vis Sci 46:2147-2155.
Nusetti S, Obregon F, Quintal M, Benzo Z, Lima L (2005) Taurine and zinc modulate outgrowth from goldfish retinal explants. Neurochem Res 30:1483-1492.
Oancea E, Meyer T (1998) Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell 95:307-318.
Okazaki Y, Morimoto T, Sawai H (2008) Parameters of optic nerve electrical stimulation affecting neuroprotection of axotomized retinal ganglion cells in adult rats. Neurosci Res 61:129-135.
Patel N, Poo MM (1982) Orientation of neurite growth by extracellular electric fields. J Neurosci 2:483-496.
Patel NB, Poo MM (1984) Perturbation of the direction of neurite growth by pulsed and focal electric fields. J Neurosci 4:2939-2947.
Qiu J, Cai D, Dai H, McAtee M, Hoffman PN, Bregman BS, Filbin MT (2002) Spinal axon regeneration induced by elevation of cyclic AMP. Neuron 34:895-903.
Rajnicek AM, Robinson KR, McCaig CD (1998) The direction of neurite growth in a weak DC electric field depends on the substratum: contributions of adhesivity and net surface charge. Dev Biol 203:412-423.
Robinson KR, Cormie P (2008) Electric field effects on human spinal injury: Is there a basis in the in vitro studies? Dev Neurobiol 68:274-280.
Sato T, Fujikado T, Lee TS, Tano Y (2008a) Direct effect of electrical stimulation on induction of brain-derived neurotrophic factor from cultured retinal Muller cells. Invest Ophthalmol Vis Sci 49:4641-4646.
Sato T, Fujikado T, Morimoto T, Matsushita K, Harada T, Tano Y (2008b) Effect of electrical stimulation on IGF-1 transcription by L-type calcium channels in cultured retinal Muller cells. Jpn J Ophthalmol 52:217-223.
Schmidt CE, Shastri VR, Vacanti JP, Langer R (1997) Stimulation of neurite outgrowth using an electrically conducting polymer. Proc Natl Acad Sci U S A 94:8948-8953.
Schnell L, Schwab ME (1990) Axonal regeneration in the rat spinal cord produced by an antibody against myelin-associated neurite growth inhibitors. Nature 343:269-272.
Song B, Zhao M, Forrester J, McCaig C (2004) Nerve regeneration and wound healing are stimulated and directed by an endogenous electrical field in vivo. J Cell Sci 117:4681-4690.
Song H, Ming G, He Z, Lehmann M, McKerracher L, Tessier-Lavigne M, Poo M (1998) Conversion of neuronal growth cone responses from repulsion to attraction by cyclic nucleotides. Science 281:1515-1518.
Spitzer NC (2006) Electrical activity in early neuronal development. Nature 444:707-712.
Tabata T, Olivera BM, Ishida AT (1996) Omega-conotoxin-MVIID blocks an omega-conotoxin-GVIA-sensitive, high-threshold Ca2+ current in fish retinal ganglion cells. Neuropharmacology 35:633-636.
Tagami Y, Kurimoto T, Miyoshi T, Morimoto T, Sawai H, Mimura O (2009) Axonal regeneration induced by repetitive electrical stimulation of crushed optic nerve in adult rats. Jpn J Ophthalmol 53:257-266.
Tang F, Dent EW, Kalil K (2003) Spontaneous calcium transients in developing cortical neurons regulate axon outgrowth. J Neurosci 23:927-936.
Tello F. (1907) La regeneration dans les voies optiques. Trab. Lab Invest. Biol. 5, 2327-248.
Torreano PJ, Cohan CS (1997) Electrically induced changes in Ca2+ in Helisoma neurons: regional and neuron-specific differences and implications for neurite outgrowth. J Neurobiol 32:150-162.
Udina E, Furey M, Busch S, Silver J, Gordon T, Fouad K (2008) Electrical stimulation of intact peripheral sensory axons in rats promotes outgrowth of their central projections. Exp Neurol 210:238-247.
Vasilcanu D, Girnita A, Girnita L, Vasilcanu R, Axelson M, Larsson O (2004) The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway. Oncogene 23:7854-7862.
Wen Z, Guirland C, Ming GL, Zheng JQ (2004) A CaMKII/calcineurin switch controls the direction of Ca2+-dependent growth cone guidance. Neuron 43:835-846.
Zhang LI, Poo MM (2001) Electrical activity and development of neural circuits. Nat Neurosci 4 Suppl:1207-1214.
Zheng JQ (2000) Turning of nerve growth cones induced by localized increases in intracellular calcium ions. Nature 403:89-93.