Dissertation submitted to the Combined Faculties for the Natural Sciences and Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Ioana Goganau born in Craiova, Romania Oral examination: 19th of September, 2016 Electrical stimulation and activity for axonal regeneration Referees: Prof. Dr. Hilmar Bading Prof. Dr. Armin Blesch PhD Thesis Ioana Goganau Table of content TABLE OF CONTENT ........................................................................................... I LIST OF FIGURES .............................................................................................. VI LIST OF TABLES .............................................................................................. VIII ABBREVIATIONS AND ACRONYMS ................................................................. IX DEDICATION....................................................................................................... XI ACKNOWLEDGEMENTS .................................................................................. XII ABSTRACT OF THE DISSERTATION ................................................................ 1 ZUSAMMENFASSUNG DER DISSERTATION.................................................... 3 1. INTRODUCTION ............................................................................................ 6 1.1. Spinal cord injury ............................................................................................................... 6 1.1.1. Incidence and outcomes of spinal cord injury............................................................... 6 1.1.2. Pathophysiology of spinal cord injury ........................................................................... 7 1.1.3. Therapeutic and experimental approaches to SCI ..................................................... 10 1.1.4. Clinical translation of SCI experimental approaches .................................................. 15 1.2. Somatosensory primary neurons, regeneration and spinal cord injury ..................... 17 1.2.1. Gross anatomy of primary sensory neurons............................................................... 17 1.2.2. Regeneration and axotomy response after peripheral lesion in DRGs ...................... 18 1.2.3. Sensory function and regeneration after SCI ............................................................. 20 1.3. Activity and neuronal regeneration ................................................................................ 23 1.3.1. Activity plays multiple roles in developing and adult neurons .................................... 23 1.3.2. Calcium/calmodulin signaling is involved in activity-dependent effects on survival and growth .................................................................................................................................... 25 i PhD Thesis Ioana Goganau 1.3.3. Acute electrophysiological response to axotomy and calcium signaling are involved in initiation of regeneration ............................................................................................................. 27 1.3.4. Peripheral but not central axotomy induces long term changes in electrical proprieties of the neurons ............................................................................................................................ 29 1.3.5. Calcium signaling and regeneration of DRG neurons................................................ 30 1.4. Electrical stimulation for axonal regeneration .............................................................. 33 1.4.1. Means of modulating neuronal activity ....................................................................... 33 1.4.2. Electrical stimulation for peripheral regeneration ....................................................... 34 1.4.3. Electrical stimulation to enhance central regeneration of primary sensory neurons . 35 References .................................................................................................................................... 38 2. RATIONALE AND HYPOTHESES ............................................................... 52 3. MATERIALS AND METHODS ..................................................................... 54 3.1. Surgical procedures ........................................................................................................ 54 3.1.1. Animals ....................................................................................................................... 54 3.1.2. Anesthesia and analgesia .......................................................................................... 54 3.1.3. In vivo electrical stimulation ....................................................................................... 54 3.1.4. In vivo growth after central lesion and cell injection ................................................... 56 3.1.5. In vivo injection of AAV in lumbar DRGs and CL in virus injected animals................ 58 3.2. Assessment of regeneration in vivo .............................................................................. 59 3.2.1. In vivo regeneration after DCL ................................................................................... 59 3.2.2. Assessment of in vivo peripheral regeneration after CL ............................................ 62 3.3. Sensory testing ................................................................................................................ 63 3.3.1. Fine touch - von Frey filaments sensory test ............................................................. 63 3.3.2. Thermal sensitivity – Plantar Heater test (Hargreaves) ............................................. 64 3.3.3. Data analysis .............................................................................................................. 64 3.4. DRG culture experimental procedures .......................................................................... 64 3.4.1. DRGs isolation ........................................................................................................... 64 3.4.2. DRGs cell culture - plates and growth medium .......................................................... 65 3.4.3. In vitro electroporation ................................................................................................ 66 3.4.4. Replating of DRGs neuron cultures ........................................................................... 66 3.4.5. In vitro depolarization ................................................................................................. 67 3.4.6. DRGs culture immunohistochemistry ......................................................................... 67 3.4.7. Neurite growth assessment ........................................................................................ 70 3.4.8. Calcium imaging in DRG cultures .............................................................................. 71 ii PhD Thesis Ioana Goganau 3.4.9. Quantification of HDAC5-FLAG export ....................................................................... 72 3.5. Differential gene expression by RNA sequencing ........................................................ 72 3.5.1. Experimental groups ................................................................................................... 72 3.5.2. DRG isolation .............................................................................................................. 73 3.5.3. RNA isolation .............................................................................................................. 73 3.5.4. Libraries and data acquisition ..................................................................................... 73 3.5.5. Data analysis .............................................................................................................. 73 3.6. Plasmids ............................................................................................................................ 75 3.6.1. List of plasmids and brief description ......................................................................... 75 3.6.2. Growing and collecting bacterial cultures for a plasmid prep ..................................... 75 3.6.3. Plasmid isolation ......................................................................................................... 76 3.6.4. Quality check of plasmids ........................................................................................... 76 3.6.5. Transforming bacteria ................................................................................................. 77 3.7. BMSCs isolation and culture for cell transplantation ................................................... 77 3.8. General statistics and data analysis ............................................................................... 78 References .................................................................................................................................... 79 4. RESULTS..................................................................................................... 80 4.1. Ex-vivo effects of in vivo electrical stimulation ............................................................ 80 4.1.1. Electrical stimulation parameters and set-up – optimization of protocol .................... 80 4.1.2. Cuff implants can potentially injure the nerve and create confounding results .......... 81 4.1.3. Hind limb movement during the stimulation is not an indicator for ES induced effects on