The dorsal column lesion model and quantification of regeneration of the injured ascending sensory afferents after experimental intervention by Mike van Zwieten (10159029) A thesis submitted for the degree of Master of Science in Brain and Cognitive Sciences Track: Behavioural Neuroscience In collaboration with Callan L. Attwell, MSc, Dr. Matthew R. J. Mason and Prof. dr. Joost Verhaagen Netherlands Institute for Neuroscience, Neuroregeneration Labarotory Co-assessor: Prof. dr. Paul J. Lucassen Swammerdam Institute for Life Sciences, University of Amsterdam Acknowledgements I would like to express my deepest gratitude to Callan Attwell, with whom I have worked with for the past 4 months. His continuous guidance has greatly transformed my way of approaching research questions, conceptional knowledge and writing scientific reports. Callan always found time to discuss with me about my questions and ideas in detail. I cannot thank him enough for his supervision, encouragement and support. I also want to thank prof. dr. Joost Verhaagen for his hospitality of taking me up in the Neuroregeneration group at the Netherlands Institute for Neuroscience and, together with dr. Matthew Mason, for the guidance, feedback and input in my project during the discussions in our weekly meetings. 2 Abstract Trauma to the spinal cord usually leads to permanent loss or severe impairment of motor, sensory and autonomic function. The lack of a pro-regenerative response in the central nervous system partially determines the irreversible structural and functional impairments following injury of the spinal cord. Experimental models for spinal cord injury, such as the dorsal column lesion model, allow the study of axon regeneration upon therapeutic interventions to promote axonal outgrowth and plasticity in injured sensory dorsal root ganglion neurons. The dorsal root ganglion neurons bifurcate into two branches, one going into the periphery and the other going into the spinal cord. Specifically, the dorsal column lesion model is characterized by its uniqueness in terms of this anatomical feature of dorsal root ganglion neurons, since the regenerative capacity of both branches fundamentally differ. The peripheral branch of the dorsal root ganglion neuron, as seen in the sciatic nerve, exhibit greater regenerative abilities compared to the central branch, which carries sensory information into the spinal cord via the dorsal columns. Altogether, the dorsal column lesion model provides an excellent model to study genetic and molecular factors underlying processes which regulate neuroregeneration. In this thesis, I will discuss the dorsal column lesion model and the variation in previous research on the level of surgical procedures, histology and functional testing. In addition, I will also elaborate on the applicability of experimental interventions in the dorsal column lesion model to promote axon regeneration and functional recovery after spinal cord injury. Glossary AAV = adeno-associated virus DRG = dorsal root ganglion Arg1 = arginase 1 GAP43 = growth associated protein 43 ATF3 = activating transcription factor 3 GCaMP = GFP calmodulin peptide BDA = biotinylated dextran amide GFAP = glial fibrillary acidic protein BDNF = brain derived neurotrophic factor GFP = green fluorescent protein B-HRP = choleragenoid horseradish peroxidase HDAC = histone deacetylase BMP = bone morphogenic protein HRP = horseradish peroxidase BMSC = bone marrow stromal cell IL-6 = interleukin 6 cAMP = cyclic adenosine monophosphate MAG = myelin-associated glycoprotein CAP23 = brain abundant membrane attached signal protein 1 OEC = olfactory ensheathing cell ChABC = chondroitinase ABC PCAF = p300/CBP-associated factor c-jun = transcription factor AP-1 PKC = protein kinase C, CL = conditioning lesion PNS = peripheral nervous system CNS = central nervous system RAG = regeneration associated gene CREB = cAMP responsive element binding protein SCI = spinal cord injury CST = corticospinal tract SLPI = secretory leukocyte protease inhibitor CTB = cholera toxin b subunit Smad1 = mothers against decapentaplegic homolog 1 dbcAMP = dibutyryl cAMP, SN = sciatic nerve DC = dorsal column STAT3 = signal transducer and activator of transcription 3 DexTR = Texas red-conjugated dextran TF = transcription factor DREZ = dorsal root entry zone YFP = yellow fluorescent protein 3 Table of contents 1 Introduction . 5 1.1 The dorsal column lesion model . 5 1.2 Thesis overview . 6 2 Experimental model for dorsal column injury . 7 2.1 Transection injury models . 7 2.2 Contusion injury models . 9 2.3 Crush injury models . 9 3 Assessment of regeneration and functional recovery . 11 3.1 Histological quantification of axon regeneration . 11 3.2 Functional analysis of regeneration . 12 4 Experimental intervention strategies to promote axon plasticity and regeneration . 14 4.1 The conditioning lesion effect and the RAG program . 14 4.2 Retrograde signaling after injury . 15 4.3 Initiation of pro-regenerative gene expression . 16 4.4 Studies on the RAG program in injured DRG neurons . 16 4.5 Regeneration-associated transcription factors . 16 4.6 Neuron-extrinsic experimental interventions . 18 5 Conclusion . 32 4 Chapter 1 Introduction Spinal cord injury (SCI) usually leads to permanent loss or severe impairment of motor, sensory and autonomic function. Although patients show some spontaneous recovery after injury, most of the patients with significant spinal cord damage are afflicted with a significant physical, emotional, social and economic burden (NSCISC 2013). Published reports showed that the annual SCI incidence rate in the US is on average 40 per million and in Europe varying from 12.1 per million in the Netherlands and 57.8 per million in Portugal (Devivo, 2012; van den Berg et al., 2010). So far management of SCI is challenging and there has been no treatment for it. This provides support for the notion that it is important to explore effective treatments for SCI patients. Most spinal cord lesions are incomplete, and in many cases, the clinical deficits reflect the extent of the injury. Depending on the nature and the severity of the injury, a spinal cord lesion could directly damage spinal axons of ascending and descending fiber tracts. Damage to these connections leaves spinal segments caudal to the lesion site partially or totally isolated from the brain (Bradbury and McMahon, 2006). The distal segment of the injured axon is retracted from the postsynaptic neuron and eliminated by degeneration (Wang et al., 2012a). The neuron together with the proximal axon segment, which usually retract a certain distance from the lesion site, typically survives, but must regenerate and reconnect to its target neurons to restore sensory function (Bradbury and McMahon, 2006). 1.1 The dorsal column lesion model Animal models are used to help predict the functional outcomes of neurological disorders and injuries and to test the efficacy of therapeutic interventions. SCI has been studied using several different animal models, including transient compression and contusion injury, complete and partial transections, crush injury and transections of specific spinal cord tracts (Figure 1 and 2). In this thesis, I focus on the technical variability in injury models of the ascending sensory neurons in the dorsal columns (DC) of the spinal cord. The DC lesion paradigm is an useful model to study axon regeneration and/or functional recovery of ascending sensory afferents following injury of the spinal cord. Sensory information that is carried in the DC arises from the periphery via dorsal root ganglion (DRG) cells. DRG neurons, often called primary sensory neurons, are pseudo-unipolar cells with a peripheral branch, such as found in the sciatic nerve (SN), and a central branch in the spinal cord, both departing from a single cell body in the DRG (Figure 3). The peripheral branch innervates peripheral targets and receives somatic information such as discriminatory touch, vibration, position sense, movement sense, conscious proprioception and tactile information (Sengul and Watson, 2014), and connect to the spinal cord via the dorsal roots. Besides that, axons in the peripheral nervous system (PNS) also derive from the autonomic nervous system for involuntary control of visceral functions (Sengul and Watson, 2014). The central branch of the DRG neurons carries this sensory information into the central nervous system (CNS) by entering the spinal cord via the dorsal root entry zone (DREZ) and ascending via the DC to subsequently connect to second-order neurons in nuclei located along the spinal cord and brain stem which project to higher centers in the brain (Figure 3). The peripheral and the central branch of DRG neurons differ fundamentally in their response to injury, even though they originate from the same cell body. The peripheral branches of DRG neurons show some degree of spontaneous regeneration after injury, whereas the central branches display very limited regeneration in response to a lesion of the dorsal roots and roughly no regeneration upon injury of the DC. The fact that central branches of DRG 5 neurons show limited spontaneous regeneration is likely due to neuron-extrinsic and neuron-intrinsic factors. After injury of the peripheral branch of the DRG neurons, a neuron-intrinsic response is initiated where expression of genes is altered towards a more pro-regenerative state, usually referred to as the regeneration-associated gene (RAG) program (Tetzlaff et al., 1991; Skene, 1989). DC axons, i.e. central branches