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 of DRG neurons, do not elicit such a regenerative response after injury. Additionally, the failure of successful regeneration of central branches of DRG neurons after injury is also partially caused by the unfavorable environment for growth at the lesion site, due to the presence of glial barriers (e.g. presence of astrocytes or accumulation of GFAP; glial fibrillary acidic protein) and inhibitory extracellular molecules (e.g. proteoglycans and myelin; see Filbin, 2003 for review) that are present in scar tissue (Silver et al., 2015). Also, the presence of certain macrophages around the lesion site, as part of the inflammatory response, limits the repair of CNS injury (Kigerl et al., 2009). Taken together, the hostile environment and the lack of activating the RAG program after injury play both a considerable role in the lack of eliciting successful regeneration in DC axons.
The different growth capacities of the peripheral and the central branches of DRG neurons make DRG neurons a commonly used model to acquire insight in the intrinsic molecular mechanisms underlying successful axon regeneration. More interestingly, the intrinsic growth state of the DC axons, i.e. the central processes of DRG neurons, can be partially enhanced upon injury by a lesion of the peripheral branch, often referred to as a ‘conditioning lesion’ (Richardson and Issa, 1984; Neumann and Woolf, 1999; for review see Hoffman, 2010). A conditioning lesion (CL) of the peripheral nerve allows a limited degree of axon regeneration of ascending sensory afferents in the DC after a spinal cord lesion. CLs are often implemented prior to a DC lesion, together with a transplantation of the peripheral nerve into the lesion site to mitigate the inhibitory environment of the CNS, resulting in regeneration into the peripheral nerve graft (Richardson and Issa, 1984; Oudega et al., 1994). This discovery opened up a novel line of research, wherein researchers investigated either the promoted intrinsic growth state for potential molecular interventions, the manipulation of the inhibitory environment of the CNS, or a combination of both. The unique properties of the DRG neuron, used in conjunction with a DC lesion, provide an excellent model to study genetic and molecular factors underlying processes which regulate neuroregeneration.
1.2 Thesis overview
The outline of the thesis is as followed:
• In Chapter 2, I introduce the DC lesion as a model for SCI to study axon regeneration of ascending sensory afferents and discuss several different techniques for the model. • In Chapter 3, I elaborate on the histological and functional assessment methods for axon regeneration or remodeling and functional recovery testing methods applicable to the DC lesion model. • In Chapter 4, I discuss experimental neuron-intrinsic and neuron-extrinsic (less extensive) strategies to promote axonal outgrowth and plasticity in injured sensory DRG neurons. • In Chapter 5, I summarise the main findings in the thesis and reflect on the importance of the DC lesion model in neuroregenerative research for SCI. I also propose several prospective directions of exploration for future research.
6
Chapter 2
Experimental models for dorsal column injury
Animal models have been developed with the aim of recreating features of the axonal and glial response to SCI. All SCI paradigms significantly improve our knowledge about injury of the spinal cord, and can provide a clinically relevant platform for developing and evaluating therapies in SCI. Depending on the focus of the study, the lesion model should be suitable to evaluate the parameters under investigation, such as assessment of regenerated DC fibers with histological tracers or the analysis of sensory recovery using functional tests (discussed in Chapter 3). The differential regenerative growth capacity of the peripheral and central branch of DRG neurons makes these neurons very useful to study the effect of experimental interventions, which aim to manipulate the intrinsic growth state or mitigate the exterior of DC axons upon injury. The DC lesion model is an experimental SCI model which allows quantitative evaluation of regenerated ascending sensory afferents and functional analysis of recovery.
The primary sensory afferents of the DC carry information regarding discriminatory touch, vibration, position sense, movement sense, conscious proprioception and tactile information to higher centers in the CNS (Sengul and Watson, 2014). As the spinal cord is segmentally organized, lesioning the DC will result in loss of sensory function depending on the level and the severity of the injury. As can be seen in Table 1 and 2, the DC lesion has been done at different levels and with different techniques. Besides differences in the lesioning techniques that have been used, where contusion, crush and transection lesions are most common (illustrated in Figure 1), there is also variation within each technique, such as the level, completeness, depth, use of bridging or grafting and closure of the lesion. Additional factors that can affect the assessment of the regenerative response include the animal species, age, functional test, survival time and type of histology and quantification methodology. Altogether, this results in ambiguity in interpretation of axon regeneration, plasticity and functional recovery.
2.1 Transection injury models
The most commonly used lesion technique in experimental SCI of the ascending sensory afferents in the DC consists of transection of the tract or hemisection of the spinal cord, which is an incomplete lesion and can be executed bilaterally lesioning the dorsal side of the spinal cord or unilaterally lesioning one complete side of the spinal cord (Figure 2A-C). Lesioning the DC by transection results in targeted interruption of the tract without complete interruption of the spinal cord, which minimizes the physical damage from the injury to other spinal cord tracts (Steward et al., 2003). Transection of the DC and hemisection of the spinal cord has been performed in many ways, as summarized in Table 1 and 2. Following spinal column exposure, laminectomy and removal of the dura, transection techniques can be carried out at different levels of the spinal cord using different sets of surgical tools, and transecting the DC axons either bilaterally or unilaterally with various postinjury treatments of the lesion. For instance, in the lab of Izthak Fischer in Philadelphia, USA they used 30-gauge needles to unilaterally severe the DC tract on the cervical level of the spinal cord, followed by excision of a small part of the DC using light suction and subsequently fill the lesion site with a GRP (glial-restricted precursors) cell graft that, besides expresses several neurotrophic factors as therapeutic intervention, provides an extracellular matrix for physical support of the injured tract (Bonner et al., 2011; Haas et al., 2012). Many other studies applied different transection approaches without any grafting, including dorsal
7 Figure 1: The most common rodent DC lesion models for SCI. A schematic diagram of the rat spinal cord and common DC lesion paradigms for SCI indicated with the striped areas, together with the instruments used to perform the lesion. (A-C) Transection injuries of the spinal cord, illustrating in (A) dorsal hemisection of the spinal cord, (B) lateral hemisection of the spinal cord and (C) bilateral transection of the DC using various sets of tools (here illustrated with the microscissors and the scalpel, all summarized in Table 1). Besides transection, these DC lesion paradigms can also be made using forceps creating a crush injury. (D) Contusion injury by dropping a weight on the spinal cord in a controlled manner. (E) Severing individual superficial DC axons using two-photon laser. hemisection at the lower thoracic level using a microknife (Tedeschi et al., 2016) or at the cervical level with a 3.8- mm-wide piece of razor blade (Onifer et al., 2005), lateral hemisection at T11-12 level of the spinal cord using microscissors (Tang et al., 2015) and DC transection at the high cervical level with a Scouten wire-knife (Hollis et al., 2015) or at the thoracic level using tungsten steel microscissors (Neumann and Woolf, 1999) or irridectomy scissors (Qiu et al., 2002). Some paradigms included making holes in advance with, for instance, a 30-gauge needle with subsequent enlargement of the holes with a 27-gauge needle followed by a cut (Fagoe et al., 2016), where others directly performed a DC transection without laminectomy (Moon et al., 2006). Additionally, the DC can also be cut to various depths, varying from superficially cutting individual DC axons (Figure 2F) using Vannas spring scissors (Ertϋrk et al., 2007; He et al., 2016) or two-photon laser cutting (Ylera et al., 2009) to bilateral complete DC transection up until the central canal (Neumann and Woolf, 1999), also severing the descending corticospinal tract (CST) in rats. Naturally, transection models create a space between the distal and the proximal portions of the spinal cord after cutting. Some experiments include filling this space with bone marrow stromal cell (BMSCs; Hollis et al., 2015), peripheral nerve implants when combined with a CL (Richardson and Issa, 1984), olfactory ensheathing cells (OECs; Toft et al., 2007), or other implants, where others do not. Although injuries like these are rarely seen in humans SCI, this technique allows one to answer questions regarding sprouting, dieback and remodeling on an axonal level (Talac et al., 2004; see for review Brösamle and Huber, 2007). All in all, transection injuries allow DC tract interruption with high confidence, establishing an isolated model to study regeneration of ascending sensory afferents in the spinal cord.
8 2.2 Contusion injury models
Besides transection models, transient compression or contusion lesion techniques are generally used to mimic a more clinically relevant situation and can be applied to study secondary injury processes or for the development of translational therapies. In a contusion lesion paradigm (Figure 2E), it is difficult to ensure the completeness of the DC lesion, which leads to higher probability of spared fibers. Since contusion models are closer to clinical cases, this technique can be used to validate transection studies. Contusion injuries can be employed in a controlled and reproducible manner due to the development of several devices. Using the Infinite Horizon Impactor device one can, after surgical exposure of the spinal cord and leaving the dura mater intact, injure the spinal cord with a controlled impact force of 225 kdyne (James et al., 2015), 200 kdyne (Soderblom et al., 2015), 150 kdyne (Baker et al., 2007) or 50 kdyne (Bartus et al., 2016) at C5, T8, T9 and T10/11 level of the spinal cord respectively. Another device is the MASCIS weight drop device that Pearse and colleagues used to make a moderate contusion injury by dropping a 10.0 g rod from a height of 12.5 mm at the midthoracic level of the spinal cord (Pearse et al., 2007). The transient force that these devices produce on the dorsal spinal cord is decided by the investigator, just as the level of the spinal cord. Cervical contusion injuries are usually to study motor dysfunction, whereas pain is more suitable to study following contusion injury at the thoracic level of the spinal cord (Gensel et al., 2006). However, contusion injuries are usually thoracic, because high cervical contusion injuries are often fatal due to the additional impact on the lungs and heart. This approach allows reliable comparison within experimental animals of the same study, but the technical variability in the contusion paradigms complicates comparison between studies. In conclusion, contusion injuries are more challenging for one to determine if the enhanced functional recovery is due to a pro-regenerative response of the injured axons or due to spared axons by the lesion. For this reason, contusion can be used to study for instance secondary injury responses, because the injury itself is closer to human SCI, or to validate transection injury models, since one can correlate the axon regeneration with the improvements in sensory function more reliably in those.
Figure 2. Detailed illustration of DC lesion models for SCI. Schematic drawings of transverse sections of adult rat cervical (C7) spinal cords (modified from Watson, Paxinos, Kayalioglu and Heise, 2015) depicted in the striped areas the injuries to the spinal cord; with (A) dorsal hemisection of the spinal cord, (B) lateral hemisection of the spinal cord, (C) complete bilateral DC transection, (D) bilateral DC aspiration, (E) spinal cord contusion and (F) single DC axon transection injuries.
2.3 Crush injury models
Another paradigm that is often used for SCI models are crush injuries, resembling a more clinical injury when compared to transection models but less so than contusion models. Usually crush injuries are performed by making holes with a very sharp instrument before a pair of forceps are inserted and closed for a certain amount of time to crush the DC while keeping the structural organization of the cord largely intact. Residual damaged tissue prevents the formation of cysts and may allow a substrate for regenerating axons to traverse. The forceps can be inserted with a
9 variable width, depth, duration and level of the spinal cord, again resulting in high variability in the employment of the lesion. A paradigm that is commonly used is the one from Bradbury and colleagues, where they inserted a pair of fine watchmakers’ forceps to a depth of 2 mm bilaterally into the spinal cord and then held tightly for 10 s before raising them back out of the spinal cord (Bradbury et al., 1999). Similar approaches for crush injuries with slightly different decisions on some technical aspects like mentioned before are listed in Table 1. For instance, Puttagunta and colleagues chose to make 2 s long crush injuries with no. 5 forceps to a depth of 2 mm and Gaudet and colleagues went for a more specific and severe paradigm where they only injured axons to a depth of 0.6 mm and held a pair of forceps tightly together for 10 s and repeated this three times (Puttagunta et al., 2014; Gaudet et al., 2016). Crush injuries leave the structural organization of the DC tract mainly intact while severing the axons, which could provide the correct physical environment for injured axons for re-growth. However, a crush injury cannot ensure completeness of the lesion and is therefore less suitable for correlation studies on axon regeneration and recovery of sensory function of DRG neurons.
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Chapter 3
Assessment of regeneration and functional recovery
3.1 Histological quantification of axon regeneration
The variation in experimental models for animal SCI demands the need for unambiguous definition of the outcome. The widely accepted view on functional recovery after SCI is that it requires robust axonal regeneration, particularly elongation. A wide range of tools is currently available to visualize neurons and their axonal projections in live animals and in processed tissue. The histological assessment of axonal retraction or outgrowth around the lesion is often accompanied with GFAP staining to determine the borders of the lesion site, which is essential to provide a quantitative perspective on elongation of regenerative axons. Regeneration can be determined with confidence when all the axons of a projecting tract are lesioned, i.e. no axons are spared, and growth of labeled axons from an identified source is observed penetrating or sprouting around the lesion site. Usually, this involves tract tracing to identify the origin, course, and termination of axons. The unique anatomical structure of DRG neurons allows transganglionic labeling by injection of neuroanatomical tracers, such as horseradish peroxidase (HRP; Richardson and Issa, 1984), choleragenoid HRP (B-HRP; Neumann and Woolf, 1999), biotinylated dextran amine (BDA; Toft et al., 2007), Texas Red-conjugated Dextran (DexTR; Parikh et al., 2011), Microruby (Puttagunta et al., 2015) and cholera toxin B subunit (CTB; Bradbury et al., 1999), into the (SN). CTB is the most commonly used tracer, ensuring high proportional labeling of DC axons by binding to the GM1 ganglioside (monosialoganglioside 1; Lai et al., 2015). Sectioning of the brainstem allows for staining of the gracile and cuneate nuclei, the point of termination of the long projecting DC afferents (Figure 3), for axons which were spared by an incomplete DC lesion - data of which need to be omitted from functional recovery analysis.
Alternatively, the expression of fluorescent proteins or GCaMP (GFP calmodulin peptide; calcium indicator) in DRG neurons prevents doing additional immunohistological procedures in tissue sections or following clearing of tissue blocs, it allows 3-dimensional imaging. Moreover, in a small number of studies live imaging has been used to study axonal regeneration in vivo. For regeneration studies, the expression of the fluorescent protein in DRG neurons allows the visualization and quantification of axons. Farnesylated eGFP (eGFPf) is a modified, membrane-bound form of eGFP that very efficiently labels intact, injured and regenerating fibers (Fagoe et al., 2014a). Methods that can introduce these molecules into the DRG neurons include electroporation of an expressing plasmid (Saijilafu et al., 2011) or viral vector injection into the DRG (Andrews et al., 2009) or SN (Gaudet et al., 2016). Alternatively, native expression of fluorescent reporter compounds in transgenic mouse lines, such as Thy1-GFP-M (Tedeschi et al., 2016), -YFP (He et al., 2016) or -GCaMP (Tang et al., 2015) models, prevent any conditioning effect by introducing the compound via injection into the DRG neuron. The use of fluorescent reporter proteins allows, for instance, two- photon time-lapse imaging to monitor changes in axonal networks in living mice (Dray et al., 2009; Farrar et al., 2012; Tang et al., 2015; Lorenzana et al., 2015) and 3D imaging of cleared spinal cords using 3DISCO clearing and light-sheet laser-scanning ultramicroscopy (Ertϋrk et al., 2012) or tetrahydrofuran-based tissue clearing and confocal microscopy (Soderblom et al., 2015), other clearing techniques are regularly appearing and depending on the need for immunological or chemical counterstaining post fixation they can vary in complexity, efficiency and time. These techniques are very helpful to investigate alternative regeneration pathways, not only around the lesion but in the whole spinal cord. Besides histological assessments, another approach can be in vivo magnetic resonance imaging (MRI) of contusion-injured rats to study neuropathologic changes of secondary injury (Weber et al., 2006).
11 3.2 Functional analysis of regeneration
Ultimately, recovery of sensory function is assessed as well as increases in axonal regeneration following injury, allowing correlation of the underlying regenerated neural circuitry to the functional recovery. In the end, the functional outcome in rats with SCI is the most important factor for evaluating the extent of injury and treatment efficacy. For this purpose, the DC lesion model is very useful, since a number of functional testing methods can be used to quantify the effects of experimental intervention. Usually, the clinical deficits reflect the severity of the injury and assessment of sensory function can reflect the extent of recovery upon injury. For rodents, after injury of the DC, sensory modalities including conscious proprioception, discriminatory touch, vibration and joint position and movement sense are affected (Sengul and Watson, 2014). Dorsal hemisection or contusion injuries are often accompanied by damage to the CST resulting in additional motor deficits. Also, DC transection lesions result in mild functional deficits that are transient and difficult to detect (Kanagal and Muir, 2007, 2008). Thereby, a DC lesion spares other sensory spinal pathways, notably the spinothalamic pathway, so much sensory information still reaches the brain. This requires useful tests for the ascending DC pathway that comprise tasks expected to be sensitive to these sensory functionalities (Fagoe et al., 2016). DC lesions at cervical and thoracic levels have different effects on locomotor abilities in rats (Kanagal and Muir, 2008). This might be related to both functional distinctions between the fore- and hindlimbs and to anatomical differences in the DC at different spinal levels. This has implications for the mechanisms of recovery as well as the types of behavioral tests which can be practically used to measure functional changes in different lesion models (Kanagal and Muir, 2008). In a recent study my lab showed that cervical lesions are preferred for assessment of functional recovery following DC lesion as the deficit is higher and sustained for a longer period of time in comparison to a thoracic lesion, this allows more time to assess for functional improvement following intervention (Fagoe et al., 2016).
As can be observed in Table 1, only a small number of regeneration studies that use the DC lesion as experimental model for SCI conduct assessment of sensory recovery, either via functional or electrophysiological testing. There are several functional tests that were developed for complex sensorimotor SCI’s, such as, one of the most classical tests, the BBB (Basso, Beattie and Bresnahan) and BMS (Basso Mouse Scale) tests for rats and mice respectively (Basso et al., 1995; Basso et al., 2006). Improvements in locomotion can also be assessed using footprint and gait analysis, either manually or using semi-automated analysis (de Medinaceli et al., 1982; Hamers et al., 2001) and muscle strength can be evaluated by inclined plane tasks or tests testing forelimb grip strength (Gale et al., 1985; Pearse et al., 2005). However, assessment of primary sensory function such as proprioceptive function is not the primary focus of these tests, and they are predominantly used with lesion models with additional damage of descending motor pathways, such as dorsal hemisection (Merkler et al., 2001; Blesch and Tuszynski, 2003; Pearse et al., 2007; Tang et al., 2015) or contusion injury (James et al., 2015).
Several other studies employ sensory tests that are primarily developed for nociceptive functions and were used to investigate perceptual abnormalities in treatment groups or neuropathic pain states as a result of the lesion (Andrews et al., 2009; Chan et al., 2005; Hollis et al., 2015). For this aim, thermal and mechanical hyperalgesia can be assessed with Hargreaves and Von Frey tests. It is arguable whether either of these tests are useful in a DC lesion as local circuitry below the lesion remains intact and unconscious-reflexive limb withdrawal may be mistaken for regeneration, so perhaps these tests should be limited to SN or dorsal root lesion where regeneration is known to occur and communication with local spinal circuitry is definitely severed or only used when a robust regenerative treatment resulting in restoration of the sensory tract is achieved and one wants to determine the presence of hyperalgesia.
The coordination of motor control from sensory feedback is essential for recovery following DC injury. Tests regarding skilled locomotion in horizontal ladder or foot slip tests (Grill et al., 1997; Kanagal and Muir, 2007; Metz and Whishaw, 2002), balance in rope or beam crossing (Kim et al., 2001; Metz et al., 1998), haptic discrimination in food reaching tasks (McKenna and Whishaw, 1999; Ballerman et al., 2001), tactile sensation in tape removal (Schallert et al., 1982) and proprioception in CatWalk gait analysis and horizontal ladder (Hamers et al., 2001; Grill et al., 1997) are fundamentally more appropriate to quantify sensory functions carried by DC axons. These tests can be used to measure overall decline after SCI, for many tests spontaneous recovery occurs within several weeks, such that
12 functional deficits become too small to detect. Recently, my lab introduced a new functional test where simultaneously tactile sensation and proprioception are evaluated in a functional testing paradigm we dubbed the ‘inclined rolling ladder’ (Fagoe et al., 2016). We showed that the inclined rolling ladder, together with the horizontal ladder and the CatWalk gait analysis, appear to be the most sensitive overall following DC transection lesion (Fagoe et al., 2016) out of the other functional tests we evaluated.
Evaluating functional recovery after SCI may be challenging by using functional testing, due to supposedly, compensatory adaptive plasticity of spared tracts. Alternatively, electrophysiological techniques provide a targeted approach to investigate the recovery of the neuronal circuitry. By stimulating peripheral targets (such as the dorsal roots) and then record cord dorsum potentials (CDPs; caudal and rostral of the lesion site) or sensory evoked potentials (SEPs; sensorimotor cortex) discloses the amplitude and latency of recorded responses (Toft et al., 2007; Bonner et al., 2011; James et al., 2015; Bartus et al., 2016), which reflects the conduction of the injured axons.
Figure 3. The anatomy of the dorsal root ganglion neuron in relationship with the peripheral nerve and the spinal cord and indicated targets for successful neuron-intrinsic experimental intervention strategies. The primary sensory DRG neurons bifurcate into two branches, one going into the periphery and the other going into the spinal cord. The central branch of the DRG neuron relays information including heat, pain and position from the body via the peripheral DRG branch and projects via the DC to the brainstem directly or indirectly via spinal neurons (not shown), such as the gracile nucleus, cuneate nucleus and the external cuneate nucleus. The panels (concurrent with the arrows) show targets for neuron-intrinsic intervention to promote axonal growth and plasticity, with (A) introduction of pharmacological agents by injection into the SN, (B) CL of the SN, (C) viral vector delivery by injection into the DRG, (D) using transgenic animals to genetically knockout or overexpress a gene of interest, and (E) subcutaneous or intrathecal injection to deliver pharmacological agents. Key: gn = gracile nucleus, cn = cuneate nucleus, ecn = external cuneate nucleus, dc = dorsal column, cst = corticospinal tract, d = dorsal nucleus (Clarke’s nucleus), rs = rubrospinal tract, lf = lateral funiculus, vf = ventral funiculus.
13
Chapter 4
Experimental intervention strategies to promote axon plasticity and regeneration
Injury to the DC leads to interruption of the tract and consequently to disruption of sensory information flow from the periphery to the brain. Usually, upon axotomy, the proximal end of axons forms a retraction bulb and the distal segment (the part disconnected from the cell body) loses its postsynaptic connection and undergoes degeneration. Regaining sensory function carried by DC axons requires robust axon regeneration of the injured axons, the synaptic reconnection to target cells and ultimately the reconstitution of a new functional neuronal circuitry. Regenerative research has provided numerous potential treatments in different SCI models. Specifically, for the DC lesion model the focus is on the regeneration/retraction via histological tracing of the injured axons or/and the functional recovery of the sensorimotor or proprioceptive tasks after treatment. In Table 1 we included the studies that employed a DC lesion together with an experimental intervention, either neuron-intrinsic (highlighted in Figure 3), neuron-extrinsic or both. Here, I examine the extent to which experimental interventions to promote axonal outgrowth and plasticity following lesion of the DC has proved valuable.
4.1 The conditioning lesion effect and the RAG program
In contrast to peripheral DRG axons, injured central DRG axons have a limited ability to regenerate. Over the last decades our mechanistic view on the disparity between the regenerative capacities in the PNS and the CNS has noticeably progressed. Besides extracellular environmental challenges, the lack of an intrinsic growth capacity largely accounts for the failure of adult central DRG axons to regenerate. After injury of the peripheral branch of the DRG axons, 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. On the contrary, injury to central branches of DRG axons result in a response in the injured neurons that is fundamentally different from the response to a peripheral lesion. Following a lesion to a central axon no RAG program is induced. However, it was discovered that implantation of a peripheral nerve graft into the spinal cord resulted in the regrowth of some central DRG axons into the graft (David and Aguayo, 1981; Richardson et al., 1982), indicating that certain central axons do contain the intrinsic capacity to regenerate when provided with a suitable growth-stimulated environment (Bray et al., 1987). Interestingly, preconditioning the peripheral branch of DRG neurons prior to a lesion of the DC, increases the intrinsic growth state of their central axons in the DC, resulting in significant regeneration into the lesion site and even beyond (Richardson and Issa, 1984; Neumann and Woolf, 1999). This is referred to as a ‘conditioning lesion’. The CL manifests the biggest effect in terms of axon regeneration 1 week before the SCI (Neumann and Woolf, 1999). However, lesioning the SN concurrently with the SCI manifest modest growth into the lesion site and there was absolutely no axonal growth when the SN was cut 1 or 2 weeks after the SCI (Neumann and Woolf, 1999). This indicates that the timing of the CL is critical, but this approach would have, however, no clinical utility and forces research to focus on potential postinjury experimental strategies. Interestingly, combining a SN lesion at the time of the SCI together with a similar lesion 1 week later (repriming), results in growth of damaged axons into and beyond the lesion site (Neumann et al., 2005). Altogether, the CL model holds a promising component in neuroregenerative research and is regularly used as a golden standard for measures of axon regeneration following DC injury.
14 4.2 Retrograde signaling after injury
The CL model makes one wonder how the regenerative response of two branches of the exact same cell can be so different following axotomy and what the molecular mechanism is behind this principle. Importantly, the CL effect was shown to be transcription dependent, which means that an initiation of a new gene expression program is required to acquire growth competence after axonal injury (Smith and Skene, 1997). In the PNS, calcium influx into the axon is one of the first signals caused by injury, which subsequently leads to a back-propagating action potential that travels towards the soma via activation of voltage-dependent sodium channels (Chierzi et al., 2005). Given this electrophysiological component in injury signaling, it was of interest to ask if artificial induction of activity can stimulate regeneration. Electrical stimulation enhanced axonal outgrowth in DRG neurons following a complete bilateral DC transection injury and this effect was associated with increased intracellular cAMP levels (Udina et al., 2008). Besides, early calcium waves after injury are likely to govern cell autonomous responses, such as growth cone formation and cytoskeletal rearrangement (Bradke et al., 2012), which are essential for axon regeneration, but also epigenetic changes (Cho et al., 2013). The back-propagating wave elicits activation of protein kinase Cµ (PKCµ) after entering the soma, which is followed by the nuclear export of histone deacetylase 5 (HDAC5), thereby increasing histone acetylation and activating the proregenerative transcription program (Cho et al., 2013).
Additionally, Finelli and colleagues show that pharmacological modulation of the activity of histone deacetylases using HDAC inhibitors in vivo leads to histone H4 hyperacetylation, induction of RAG expression and increased axon regeneration after DC injury in mice (Finelli et al., 2013). Furthermore, histone acetyltransferase 1 p300/CBP-associated factor (PCAF), which can form a complex with the histone acetyltransferase p300, is involved in epigenetic modification at promoters of several RAGs and activate GAP43 (growth associated protein 43, galanin and BDNF (Puttagunta et al., 2014). In addition, AAV-PCAF injection into the SN resulted in an increase in regenerated fibers into and beyond the lesion site after a DC crush injury (Puttagunta et al., 2014). These studies allude to the involvement of epigenetic changes in the regulation of the RAG program and axon regeneration in the early calcium-dependent phase of injury signaling. Moreover, the fast axonal calcium influx after injury is critical in other, later and somewhat delayed retrograde signaling pathways, which are dependent on transport of macromolecular factors that are trafficked via cellular motor complexes.
The acquisition of an increased (transcription-dependent) growth state of DRG neurons following damage to the peripheral branch indicates prior axonal retrograde transport of axon-regenerative signals to the nucleus (Smith and Skene, 1997). These include importins and JNK-associated signaling molecules (see Rishal and Fainzilber et al., 2010, 2014 for reviews on retrograde signaling in axonal regeneration). In addition, blocking the retrograde transport of such injury signals inhibits the activation of certain regeneration-associated TFs (discussed below) and prevents efficient regeneration (Ben-Yaakov et al., 2012). Logically, the supply of pro-regenerative agents towards the soma and the lesion site depends on transport and the cytoskeletal architecture. The reformation of the cytoskeletal architecture of injured central axons has been shown to be very limited, while peripheral axons form a growth cone that enables regeneration a short time after injury (Bradke et al., 2012). After an injury in the PNS, many axons reform motile growth cone-line structures within hours (Ertürk et al., 2007), whereas injured central DRG axons retract or exhibit fragmented degenerative morphologies (Lorenzana et al., 2015). Naturally, for axons to regenerate after a lesion, lengthy elongation is dependent of the supply of membranes, cytoskeleton, organelles and other building blocks in the growing tips (He and Jin, 2016). The ability of axons to form a motile growth cone-like structure may be compulsory for the regenerative process. For a treatment to work, the growth cone of the proximal end needs to be re- established, allowing regrowth and lengthy elongation resulting ultimately in connecting to other neurons. Pharmacological stabilization of microtubules after SCI, by taxol application onto the lesion site, resulted in less axonal degeneration of DC axons (Ertürk et al., 2007) and enhanced growth of Raphe-spinal tract axons, without showing its effect in sensory afferents (Hellal et al., 2011). Application of another microtubule stabilizer, epothilone B (EpoB), induced promoted axon regeneration (Ruschel et al., 2015). Remodeling of the axonal cytoskeleton may be another aspect to be addressed in a combinatorial approach to gain a full regenerative response following SCI.
15 4.3 Initiation of pro-regenerative gene expression
The fact that adult pseudo-unipolar DRG neurons have a growth-competent branch in the periphery and the other, growth-incompetent branch, in the CNS, makes these DRG neurons a uniquely relevant cell to study intrinsic growth capacity of neurons. A category of regeneration-associated factors whereof expression or activity is altered upon peripheral axonal injury, but not in central axonal injury, are involved in the cAMP/PKA/CREB pathway (see Hannila and Filbin, 2008 for a review). After a CL levels of cAMP are transiently increased in the central processes of DRG neurons (Qiu et al., 2002). Thus, cAMP is triggered by axonal injury and activates PKA that in turn phosphorylates the cAMP responsive element binding protein (CREB), required for activation of gene expression. Interestingly, interference with the cAMP/PKA/CREB pathway in the CNS can be achieved by transganglionic injection in the periphery, which is more accessible and reduces damage from the intervention. Overexpressing cAMP via transganglionic injection of dbcAMP (PKA activator) results in an increase in axonal growth near and beyond the lesion site (Qiu et al., 2002; Neumann et al., 2002), approaching the CL effect. This effect is presumably overcoming the inhibitory effect of CNS myelin proteins, like MAG and Nogo, on a molecular level. In fact, dbcAMP intervention combined with a peripheral graft (environment without CNS inhibitors) did not result in axonal elongation, supporting the notion that cAMP effectors do not increase the intrinsic growth state of CNS axons (Han et al., 2004). CREB is also one of the TFs that are found to be upregulated upon peripheral axotomy, studied with the microarray studies (discussed below). Constitutive expression of CREB promotes regeneration of lesioned DC axons (Gao et al., 2004), and also upregulates several target genes, such as arginase 1 (Arg1), interleukin (IL)-6, secretory leukocyte protease inhibitor (SLPI), and methallothionen (MT). Studies that investigated the necessity of these genes in the CL mediated regenerative effect found that the CL effect is reversed in animals with conditional ablation of IL-6 (Cao et al., 2006; Cafferty et al., 2004), SLPI (Hanilla et al., 2013) and MT (Siddiq et al., 2015). Additionally, overexpressing IL-6 in DC lesion model and SLPI and MT in an optic crush model promotes modest axon regeneration (Cao et al., 2006; Cafferty et al., 2004; Hannila et al., 2013; Siddiq et al., 2015; see for review Siddiq and Hannila, 2015). To develop effective treatments for SCI, it will be necessary to examine the combinatorial effects of these cAMP effectors to achieve a synergistic effect on axonal regeneration and functional recovery (Siddiq and Hannila, 2015).
4.4 Studies on the RAG program in injured DRG neurons
It has been shown that successful axon regeneration is correlated with the (re)expression of several regeneration- associated proteins, such as GAP43 and CAP23 (Mason et al., 2002, 2003). The regenerative capacity of DC axons might be dependent on the expression of RAGs and this created a novel line of research focusing on facilitating axon regeneration by enhancing the expression of RAGs in injured DRG neurons. The different intrinsic responses of the two branches of DRG neurons to conditioning has been extensively investigated by a number of studies employing transcriptional profiling using microarray to obtain insight in the gene and TF expression patterns underlying axon regeneration (Macgillavry et al., 2009; Stam et al., 2007; Zou et al., 2009). Analyses of these genome-wide transcriptional profiling studies confirmed the involvement of some classical RAGs and identified hundreds of new genes that are potentially part of the RAG program (see for review: Fagoe et al., 2014b; van Kesteren et al., 2011; Tedeschi et al., 2012). These microarray studies have contributed significantly in the identification of key aspects of the RAG program; however, the function of many RAGs and whether they have a direct role in axon regeneration is not known. Therefore, it is essential to carry out functional studies to identify the key RAGs.
4.5 Regeneration-associated transcription factors
Alongside the inhibitory extracellular environment of the CNS, the limited growth capacity of DC axons is a major reason for the regenerative failure in the CNS. As already mentioned, the growth state of the DRG neuron can be changed by peripheral branch axotomy through activation of transcriptional regulatory networks of hundreds of specific neuron-intrinsic RAGs. The analysis of genome-wide transcriptional profiling studies provided subsets of
16 genes that act together in regulatory networks and control this proregenerative cellular response. Interference with gene regulatory networks requires a strategic approach, since these networks are inherently robust against random perturbation (Albert et al., 2000). Previous studies have shown that the manipulation of single RAG expression levels is not sufficient to trigger robust axon regeneration following injury (see Table 1). The joint manipulation of multiple genes showed enhanced axon regeneration, as seen by overexpressing both CAP23 and GAP43 resulting in enhanced DRG regeneration into peripheral nerve grafts implanted in the spinal cord (Bomze et al., 2001). The identification of highly connected key players in these gene regulatory networks, so called ‘hub genes’, have led to more targeted approaches to orchestrate coordinated expression or activity of many other genes in the RAG program (van Kesteren et al., 2011). Hub genes, which are often TFs, are therefore promising targets for interference with the RAG program at the network level. Addressing experimental interventions to these hub RAGs or a combination of RAGs with a synergetic effect is much more effective than aiming for individual randomly chosen downstream RAGs, like for instance GAP43.
STAT3 is one TF that is upregulated upon peripheral injury and suppression or knock out leads to reduced expression of another RAG, GAP43, and to an attenuation of the conditioning effect in vivo (Qiu et al., 2005; Bareyre et al., 2011). Sustained STAT3 expression promotes collateral sprouting in the mouse DRG after a DC lesion in a rapid but short-lived manner (Bareyre et al., 2011), indicating a time-dependent role of STAT3 in the RAG program. ATF3 was found to be upregulated upon peripheral injury (Seijffers et al., 2007). In a transgenic mouse line that constitutively express ATF3 the peripheral axon regeneration was to an extent comparable to that induced by a CL (Seijffers et al., 2007). However, the central branches of DRG neurons failed to elicit such a pro-regenerative response after DC injury when constitutive ATF3 expression (Seijffers et al., 2007). Activation of Smad1, another regeneration- associated TF, by overexpression of BMP4 using AAV8 in DRG neurons, resulted in improved sprouting of DC axons after injury (Parikh et al. 2011). Other regeneration-associated TFs, which have shown to have pro-regenerative effects in a peripheral nerve injury model, include NFIL3 (MacGillavry et al., 2009), C/ EBPδ (de Heredia and Magoulas, 2013), c-Jun (Saijilafu et al., 2011), SOX11 (Jankowski et al., 2009; Jing et al., 2012), and several KLF family members in CST injury models (Blackmore et al., 2012). The overexpression of single regeneration-associated TFs to enhance the central axonal regenerative response following DC injury has not yet resulted in robust axon regeneration accompanied with functional recovery. However, keeping in mind that the microarray studies showed the extent and complexity of the RAG program and likelihood that TFs coordinate expression of different, partially overlapping sets of RAGs, suggests that of combination of regeneration-associated TFs is required to activate the full RAG program and that cooperation of multiple TFs may therefore activate a larger part of the RAG program than individual TFs.
Taken together, individual TFs may each play a specific role in the regeneration process. To activate the whole RAG program, it might be necessary to manipulate the expression of several ‘hub’ TFs simultaneously, as these ’hub’ TFs presumably fulfil a central function in regulatory networks (Fagoe et al., 2014b; van Kesteren et al., 2011). For instance, it has been shown that ATF3 and c-Jun synergize via dimerization in regenerating neurons (Seijffers et al., 2006; Pearson et al., 2003) and together with STAT3 they regulate a damage-induced neuronal endopeptidase promoter (DINE) by interacting with general TF Sp1 (Kiryu-Seo et al., 2008). The AP-1 binding site also allows multiple TFs, such as Smad family members with c-Jun, to interact, allowing activation of transcription (Zhang et al., 1998; Liberati et al., 1999; Wong et al., 1999). It has been proposed that an orchestrated sequence of transcriptional events controlling the expression of specific sets of genes may be the underlying basis of an early cell-autonomous regenerative response (reviewed in Tedeschi, 2012). Recently, we showed that the AAV-mediated delivery of a combination of up to four RAG TFs (ATF3, Smad1, c-Jun and STAT3) expressed simultaneously in a significant number of DRG neurons is technically feasible; however, while it resulted in faster axonal outgrowth in the dorsal root lesion, there was no increased regeneration after DC lesion (Fagoe et al., 2015). In addition, delivery of multiple regeneration-associated TFs was not more effective than expressing ATF3 alone in this instance (Fagoe et al., 2015). The identification of the key TFs capable of robustly promoting regeneration will require a better understanding of the full regulatory network controlling RAGs (van Kesteren et al., 2011; Chandran et al., 2016).
17 Besides TFs and epigenetic modulators, there are other regulators in the RAG program with overlapping properties. Non-TFs are upregulated as part of the RAG program involved in many functions, including genes that encode adhesion/guidance molecules, neuropeptides, structural and cytoskeletal-associated proteins, and metabolic enzymes (Ma and Willis, 2015) and influence expression of RAGs. For instance, deletion of miR-155 (micro RNA- 155-5p) in neurons results in upregulation of several neuron-intrinsic RAGs, such as SPRR1A and ATF3 (Gaudet et al., 2016). Additionally, deletion of miR-155 augments the intrinsic axonal growth and most interestingly, also attenuates inflammatory signaling in macrophages (Gaudet et al., 2016). By acting on two major aspects of neuron- extrinsic as well as the neuron-intrinsic barriers that underlies the regenerative failure of DC axons after injury, miR- 155 shows to be a novel therapeutic target for axonal repair after SCI.
4.6 Neuron-extrinsic experimental interventions
As briefly mentioned, one of the reasons why CNS axons do not show spontaneous regeneration is due to the hostile environment of the injury site, where the highly inhibitory glial scar and the presence of extracellular inhibitory proteins produce a barrier for axonal growth. The scar consists of fibroblasts and together with oligodendrocytes in the CNS they express molecules that inhibit regeneration. One of the major components in the lack of regeneration of CNS axons is the presence of myelin produced by oligodendrocytes (Caroni and Schwab, 1988), and myelin- associated inhibitors, including Nogo, MAG (myelin-associated glycoprotein) and OMgp (oligodendrocyte myelin glycoprotein). Neutralizing the inhibitory properties of CNS myelin by injecting hybridoma cells expressing monoclonal antibody IN-1 at the lesion site following a dorsal hemisection has shown functional improvement in locomotive behaviour (Merkler et al., 2001). Other studies have shown modest axon regeneration upon DC injury with transplantation approaches, such as olfactory ensheating cells (OEC; Moreno-Flores et al., 2006; Pearse et al., 2007; Toft et al., 2007; Andrews and Stelzner, 2004), bone marrow stromal cells (BMSCs; Hollis et al., 2015; Hollis and Zou, 2012), NRP/GRP (Bonner et al., 2011; Haas et al., 2012), neural stem cells (NSCs; Lu et al., 2003) or fibroblasts (Blesch and Tuszynski, 2003; Roet et al., 2013). Transplants are often modified so that they overexpress growth factors. Growth factors can also be otherwise applied (via intrathecal or viral vector delivery (Fagoe et al., 2014a) for example) into the lesion, such as NT-3 (Hou et al., 2012; Baker et al., 2007; Sayer et al., 2002), NGF (Zhang et al., 2013); GDNF and BDNF (Lu et al., 2003; see Harvey et al., 2015 for a review on neurotrophic factors in SCI). A promising neuron-extrinsic strategy is to treat the scar directly to create a more permissive environment with the enzyme Chondroitinase ABC (ChABC), which digests CSPGs (chondroitin sulphate proteoglycans), which have a significant inhibitory impact on axonal regrowth in the CNS (Bradbury et al., 2002; James et al., 2015; Grosso et al., 2014; Massey et al., 2006).
Strategies aimed at fostering neurological recovery by promoting axonal sprouting and regeneration have largely targeted the glial inhibitory environment that develops following CNS injury. However, experimental evidence suggests that providing a favorable environment may not be the sole and sufficient means for functional regeneration, and that activating the limited intrinsic potential of neurons to sprout and regenerate may represent an alternative and complementary therapeutic approach. Efforts using combinatorial manipulation of multiple factors have shown great promise for devising therapeutic strategies in functional recovery of injured axons.
18
Table 1. Overview of the studies that conducted experimental DC lesion paradigms with a description of their technical approach, the experimental intervention (E = neuron- extrinsic, I = neuron-intrinsic, C = combination of neuron-intrinsic and -extrinsic), histological and functional assessment of axon regeneration and their main results.
Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader Bilateral T8 The spinal cord was exposed at thoracic level T8 by dorsal C C1q KO, CL performed at time of Anterograde axonal tracing Change in the direction of ascending Peterson et al., Anderson, A complete DC laminectomy, followed by bilateral dorsal column transection SCI with ligature and transection using CTB (6 weeks after SCI) axon growth within the lesion after 2015 transection using marked microdissection scissors SCI after CL (mice)
Bilateral T8-9 A small hole was made in the pia on the dorsal surface of the E Immunophilin treatment via Transganglionic labeling of FK506 treatment alone promote the Bavetta et al., Anderson, P complete DC cord using a fine needle and this was enlarged across the subcutaneous administration of DC axons with CT-HRP (1- survival of ascending sensory axons, 1999 transection midline using microsurgical scissors, at the same time cutting immunophilin ligands FK506, 10 weeks after SCI) combined with CL resulted in massive (rat) the dorsal vein. After hemostasis had been achieved, a fine GPI1046 and MP and peripheral sprouting at the lesion sites and some needle (25 gauge) was swept across the dorsal aspect of the conditioning lesion regeneration, whereas GPI1046 and cord two or three times to a depth of 1.5 mm MP had no such effect
Bilateral T6-7 We used the DC injury model in the rat (Neumann and I PNL lesion (unilateral sciatic Axonal tracing using B-HRP Increased length of regenerating fibres Neuman et Basbaum complete DC Woolf, 1999). Briefly, using microscissors we transected the ligation + transection distal to (6-8 weeks after SCI) 0.6 mm beyond the lesion after 6-8 al., 2005 transection DC bilaterally at T6-T7, between the dorsolateral funiculi suture) performed at the time of weeks in repriming model only (rat) the DCL (priming) then a second PNL 1 week post DCL (repriming)
Bilateral T6-7 Using tungsten steel microscissors, we transected the dorsal I Microinjection of dbcAMP in Axonal tracing using B-HRP Increased length of regenerating fibres Neumann et Basbaum complete DC columns bilaterally at the level of the T6-7 thoracic segments lumbar dorsal root ganglia (6-8 weeks after SCI) into and beyond the lesion, ± 1.6 mm al., 2002 transection (Neumann and Woolf, 1999), between the dorsal root entry measured proximal from lesion (rat) zones, under halothane anesthesia
Bilateral T13- Lamina at the level of T13/L1 removed to expose the dorsal E Triazine compound F05 In vivo fluorescent imaging of A reduction in retraction bulbs and Usher et al., Bixby, L1 complete columns, iridectomy scissors were used to transect the dorsal application onto the lesion site GFP-M mice, (immediately increase in growth distance 2010 Lemmon and DC transection columns bilaterally immediately after injury after SCI up to 24 and 48 h) Bradke (mice)
Bilateral C3 Dorsal funiculus lesions were made at the caudal aspect of C3 E Superficial injection of tet-off LV- Transganglionic labeling with NT-3 expression resulted in Hou et al., Blesch complete DC using a David Kopf Instruments microwire device as NT-3 immediately after grafting. CTB for axonal tracing (4 regeneration of axons into and 2012 transection previously described (Lu et al., 2004; Taylor et al., 2006). The NT-3 expression was regulated weeks after SCI) beyond the lesion site. There is a (rat) wire knife was lowered into the spinal cord to a depth of 1.1 with doxycycline significant decline in axon growth mm ventral to the dorsal cord surface and 1.1 mm to the left when NT-3 expression is turned off, of the midline. The tip of the wire knife was extruded, or transiently expressed. Regenerated forming a 2.25 mm wide arc that was raised to the dorsal axons were located in white matter surface of the cord, transecting the DC and CST. Cell cavity is and did not form axodendritic grafted with BMSCs synapses, but expressed presynaptic markers
19 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader Bilateral C3 Dorsal column lesions were made at the caudal aspect of C3 E Grafting of NT-3 +/- MSCs at Axonal tracing using CTB (4 Significantly more ascending sensory Taylor et al., Blesch complete DC using a David Kopf Instruments (Tujunga, CA) microwire lesion and LV- +/-NT-3 rostral at weeks after SCI) axons extended into tissue rostral to 2006 transection (rat) device. After fixation in a spinal stereotaxic unit, a small dural the lesion, immediately after SCI lesion in animals with NT-3 vectors incision was made. The wire knife was lowered into the spinal compared with GFP cord to a depth of 1.1 mm ventral to the dorsal cord surface and 1.1 mm to the left of the midline. The tip of the wire knife was extruded, forming a 2.25-mm-wide arc that was raised to the dorsal surface of the cord, transecting the dorsal funiculus including the ascending (sensory) and descending (corticospinal) axon tracts
Bilateral T8 Rats were anaesthetized with isoflurane. T8 spinal cord E Injection of LV/NGF-sNgR Transganglionic axonal tracing Regenerating sensory axons found in Zhang et al., Bo complete DC exposed by laminectomy. Dura was opened and the dorsal (truncated, soluble Nogo receptor using CTB (6 weeks after SCI) the lesion cavity of LV/NGF-sNgR 2013 crush and column was crushed with a pair of watchmakers' forceps and 1) or LV/NGF- GFP only, rostral injected animals, few axons observed transection then cut using microsurgical scissors at the depth of 1.5 mm and caudal to the SCI in or LV/NGF- GFP only (rat) (Zhang et al., 2007)
T8 unilateral Spinal cord was exposed at T8 by laminectomy. The dura was E LV- PST injection into the lesion Transganglionic axonal tracing Axons from CL only group Zhang et al., Bo and DC transection opened using fine microsurgical scissors. The dorsal column and around. Sciatic nerve using CTB, CGRP IHC for terminated at caudal border of lesion, 2007 Richardson and bilateral was crushed with a pair of watchmakers' forceps and the left conditioning lesion by crushing nociceptive axon labeling (6 LV-PST group allowed axonal crush dorsal column was cut using microsurgical scissors at a depth weeks after SCI) penetration into and beyond the (rat) of 2 mm lesion (20 times more axons than CL only) C5 SC Animals were positioned and stabilized with the impactor E Chondroitinase gene therapy via Functional: Improved levels of conduction James et al., Bradbury contusion probe of an Infinite Horizon impactor positioned 3-4 mm intraspinal injections of LV- Electrophysiological assessment through the injury site and significant 2015 (rat) above the exposed spinal cord and subsequently delivered an ChABC around the lesion site of sensory function, horizontal functional improvements in forelimb impact force of 225 kdyne to the spinal cord, inducing a ladder test, forelimb grip- and hindlimb on horizontal ladder moderate C5 contusion injury strength and the inclined plane and forelimb grip strength task (A) T11-12 DC (A) A T11-T12 laminectomy was performed and spinal cord E (A) PGB administration (i.p.) 7 (A) chronic 2p window (A) increased axonal regeneration Tedeschi et Bradke complete crush was crushed with #5 modified forceps (Dumont, FST). The days before SCI imaging of Thy1-GFP-M mice compared to vehicle treated (which al., 2016 forceps were deliberately positioned to sever dorsal column 3 days post treatment degenerated) axons completely (7 days after first treatment injection) (postinjury)
(B) T11-12 (B) A T11-T12 laminectomy was performed. The dorsal half (B) Sustained PGB administration (B) Multiphoton imaging after (B) Regeneration comparable complete DC of the spinal cord was cut using a microknife (10316-14, Fine commencing 1 hour or 2 weeks AAV-eGFP injection into the regeneration with CL, less prominent transection Science Tools (FST)) after SCI, CL control group SN to transduce L4 & L5 when treatment starts 2 weeks DRG (2 or 4 weeks after SCI) postinjury
(C) T11-12 (C) A T11-T12 dorsal hemisection was performed using a (C) PGB administration 1 h or 2 (C) Multiphoton imaging (C) PGB increased DC regeneration dorsal microknife (10316-14, FST) weeks postinjury or PNL after AAV-eGFP injection (as compared to GFP hemisection above) 4 weeks after SCI
20 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader T12 (A) and T9 (A) Mice were deeply anaesthetized and received bilateral E (A) Injection with microtubule (A) In vivo live imaging of (A) Fewer retraction bulbs, reduced Ruschel et al., Bradke (B) Bilateral dorsal column lesion at T12 stabilizing agent, epoB at lesion site adult Thy1-GFPM mice (every axonal dieback and increased 2015 complete DC (24 h before SCI) day up to 4 days after SCI) regenerative growth transection (mice) (B) Mice were deeply anaesthetized and the spinal cord was (B) Animals received (B) Axonal tracing using (B) An increase in axon regeneration exposed by laminectomy at T9. The dorsal column was intraperitoneal (i.p.) injections of Microruby (4 weeks after SCI) (approx. 100µm regeneration beyond transected using a micro knife (Feather). epoB, 1 and 15 days after SCI) lesion)
(A) Bilateral T8 (A) Dorsal laminectomy was performed at T8 to expose the E (A) PNL (unilateral sciatic ligation (A) Axonal tracing using CTB (A) An increase in regenerated axons Ylera et al., Bradke complete DC spinal cord, and a bilateral dorsal hemisection was performed + transection distal to suture) 2 (6 weeks after fresh SCI) in pre-CL & post-CL + fresh DCL 2009 transection (rat) with titanium microscissors, to completely cut the dorsal weeks post injury + fresh DCL groups, up to 1.3 mm. No columns caudal to initial DCL 3 weeks post regeneration in post-CL or only DCL injury groups.
(B) Bilateral L4- (B) Fine irridectomy scissors (FST, Heidelberg) used to (B) PNL or PNL + fresh DCL (B) In vivo imaging of GFP-M (B) Axonal growth up to hundreds of 5 DC carefully transect the central DRG axons in dorsal columns transgenic mice (up to 48 h micrometers beyond the lesion within transection close to the dorsal vein. Only surface axons targeted. post injury) 48 h post injury in PNL or PNL + (mice) fresh DCL, no regeneration in unconditioned group
(C) Single DC (C) Laser power was maintained at 30 mW for imaging and (C) PNL (before or after SCI) (C) In vivo imaging of GFP-M (C) Axonal growth up to 1 mm (14 transections 300 mW for cutting single axons mice at 0 h, 3 d, 7 d, 10 d, and days after 2-photon cut, 7 days after using 2-photon 14 d after SCI conditioning) laser (mice)
Superficial After removing the laminas at T11/12 level, a small unilateral E Taxol (a microtubule-stabilizing In vivo fluorescence imaging of A reduction in amount of retraction Ertϋrk et al., Bradke unilateral T11- lesion was applied with vannas spring scissors (FST) by drug) application onto the lesion GFP-M and YFP-H transgenic bulbs and retraction distance of 2007 12 transection transecting only the superficial axons of the dorsal columns site each hour, starting mice (immediately after SCI injured DC axons (mice) immediately after lesioning up to 6 h)
T8 SC Contusion injury was induced by the MASCIS weight drop E At 1 week postinjury, rats received Transganglionic axonal tracing No significant growth of ascending Pearse et al., Bunge contusion device (Gruner, 1992). Laminectomy at T8 exposed the dorsal fluid grafts of SCs, OEG or both using CTB by injection into sensory fibres into the graft, although 2007 (rat) surface of the spinal cord underneath without disrupting dura cell types the SN, CGRP IHC to assess more CGRP+ axons caudal to injury. mater. Exposed spinal cord moderately injured by dropping a sensory fiber growth (10 weeks The SC and OEG combination group 10.0 g rod from a height of 12.5 mm after SCI) exhibited significant improvement in Functional: BBB, footprint BBB 5-9 weeks post transplantation analysis
Bilateral T10- Bilateral dorsal column transection without laminectomy I T4 transgenic mice that Axonal tracing using CTB (4 No regrowth of ascending DC axons Moon et al., Bunge 11 complete between T10-11 gap, stainless-steel wire (31 gauge; Small overexpress tPA weeks after SCI) and no functional recovery 2006 DC transection Parts Inc., Miami Lakes, FL) bent to 90° 1 mm from one end Functional: Rotarod (mice) was inserted vertically to a depth of 1 mm and moved four times across the lateral extent of the keyhole
21 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader T9-10 complete A T9-10 laminectomy was performed and the dorsal half of I AAV-PCAF injection in the left Axonal tracing using PCAF KO mice show no CL effect. Puttagunta et Di Giovanni DC crush the spinal cord was crushed with no. 5 forceps (Dumont, Fine SN (2 weeks before SCI) and Microruby 2 weeks before PCAF overexpression leads to an al., 2014 (mice) Science Tools) for 2 s. The forceps were deliberately PCAF KO + CL (1 week perfusion (perfused 6 weeks increase in regenerating fibres across positioned to sever the dorsal column axons completely preconditioning sciatic “lesion”) after SCI) the lesion and up to 1 mm rostral of the lesion
Bilateral C4-5 The dura was retracted and the dorsal columns, identified I Injections of AAV-fGFP + AAV- Injection of AAV-fGFP in the An increase in sprouting axons within Andrews et al., Fawcett DC crush within the immediate extents of the dorsal root entry zones on α9 integrin in the DRGs directly DRGs directly after SCI allows the lesion site and withdrawal 2009 (rat) either side, were crushed bilaterally for 10 s using finely milled after SCI confocal microscopy of DRG latencies near to preoperative levels. forceps at a depth of 2 mm at level C4-5. axons (perfused 6 weeks after The pressure test did not reveal any SCI) significant difference Functional: Pressure and infra- red hot-plate test
Bilateral C4 DC Bilateral lesion to the dorsal columns at spinal level C4 E Intrathecal delivery of ChABC Axonal tracing using CTB (8- Promoted regenerative axonal Bradbury et Fawcett and crush lesion (Bradbury et al., 1999). The DC were crushed with fine (directly after injury) 10 weeks after SCI). appearance (growth cones) of al., 2002 McMahon (rat) watchmakers’ forceps (0.3 mm in width), pushed 2 mm down Functional: Tape removal test, ascending sensory projections. into the cord and held for 10 s beam walk, gridwalk, footprint Significant recovery in tape test, beam analysis and gridwalk. Stride length shorter and width wider
T8-9 DC Depth is 1 mm I MP-I/II KO + CL (1 week Axonal tracing using CTB (5 MT-I/II KO reverses CL effect. CL Siddiq et al., Filbin transection Tools not specified preconditioning unilateral sciatic weeks after SCI) animals regenerated 300 µm, wt 100 2015 (mice) “lesion”) µm
T8-10 DC Depth is 1mm I SLPI deficiency CL (1 week Axonal tracing using CTB (5 SLPI KO abolishes CL effect (~100- Hannila et al., Filbin transection Tools not specified preconditioning unilateral sciatic weeks after SCI) 200 µm) 2013 (mice) “lesion”)
Bilateral T6-7 Bilateral dorsal column lesion was performed at the T6-7 level, I Intrathecal delivery of IL-6 or CL Retrograde axonal tracing CL and IL-6 both effect in an increase Cao et al., Filbin DC transection as previously described (Qui et al., 2002) (1 week preconditioning unilateral using HRP or CTB (6 weeks) in distance of regenerated fibers closer 2006 (rat) sciatic transection) in IL-6 KO to the epicenter, and even beyond. IL- mice 6 KO does not abolish CL effect
Bilateral T6-7 A dorsal column lesion was carried out as described in Qiu et I Delivery of VP16-CREB to the L4 Transganglionic axonal tracing An increase in growth of axons up to Gao et al., Filbin DC transection al., 2002, at T6-7 DRG (4 days before SCI) with CTB (24 days after SCI) and into the lesion site. Distance of 2004 (rat) axonal growth is on average 400 µm further than controls
Bilateral T6-7 Irridectomy scissors were used to cut the dorsal columns I L5 DRG dbcAMP injection (1 Transganglionic axonal tracing A 2 mm increase in fiber length into Qiu et al., Filbin DC transection bilaterally at the thoracic level T6-8 week before SCI) with HRP (1-2 weeks after the lesion site (average of 3 longest 2002 (rat) SCI) axons)
22 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader Unilateral C4-5 A 30-gauge needle was used to make a unilateral 1-mm E GRP cell graft; GRP cells treated Axonal tracing using CTB (3 An increase of axons into but not out Haas et al., Fischer DC transection incision into the right dorsal column at the C4-5 level to sever with either FBS, BMP-4, CNTF or weeks after SCI) of the grafted area with all treatments 2012 the tracts; a small segment of the right dorsal column at the bFGF (control) during site of the incision was excised differentiation
Unilateral C1 A 30 gauge needle was used to make a complete injury in the E NRP/GRP graft, LV-BDNF Axonal tracing using CTB, and An increase of NRP/GRP axons into Bonner et al., Fischer DC transection right dorsal columns at the C1 level, severing the tract, and a injection into the DC (1 week after c-fos staining and immune- the dorsal column towards BDNF 2011 (rat) small piece of the dorsal columns was excised using light SCI) electron microscopy (6 weeks gradient,, formation of synapses with suction. Although the entire right dorsal column was severed, after SCI) host neurons and (delayed) the left dorsal column was only partially damaged Functional: conduction of electrical stimulation Electrophysiological assessment through graft of sensory axon function
Bilateral T8 Following a hemi-laminectomy at thoracic spinal level 8 I Electrical stimulation for 1 h (20 Axonal tracing using CTB (14 Electrical stimulation stimulated Udina et al., Fouad complete DC (Th8), a lesion of the dorsal funiculus was performed using Hz, 0.02 ms) to the intact SN and weeks after SCI) axons enter the lesion and grow up to 2008 transection micro-scissors. This ablated, amongst others, ascending CL (concomitant unilateral sciatic 1.5 mm, but not further than the CL (rat) sensory axons including those with axons in the sciatic nerve transection & suturing to underlying muscle)
T8 dorsal Laminectomy at T8. Using iridectomy scissors, a dorsal over- E Unilateral hippocampal injection Functional: BBB, grid walk, Improvements on all locomotor tests, Merkler et al., Fouad hemisection hemisection, sparing “just parts of the ventral funiculus” of hybridoma cells producing misstep withdrawal, narrow with accompanying EMG 2001 (rat) Nogo-a inhibitor- mAb IN-1 (up beam crossing, tail flick confirmation of improved locomotor until 7 days postinjury) (weekly assessed starting on function. No improvements in day 7 postinjury up to day 35) nociceptive tail flick test and EMG of hindlimb muscles (40 days after SCI)
T9 SC Laminectomy at T9, dura left intact. The impactor force of E Intrathecal or intraparenchymal Axonal tracing using CTB (7 Neither site of NT-3 infusion Baker et al., Hagg contusion (rat) the IH impactor (Precision Systems Instrumentation, infusion of NT-3 days after SCI) improved innervation of the gracile 2007 Lexington, KY; Scheff et al., 2003) was set at 150 kdyne and a nucleus following contusion contusion delivered. The impactor probe was placed 3 mm above the cord
Bilateral T9 Laminectomy at T9. After opening the dura mater, a 1.5-2- C Peripheral nerve grafting 1 week Axonal tracing using CTB (4 An increase in regenerated fibers that Oudega et al., Hagg complete DC mm long segment of the dorsal funiculus was removed with before or immediately after SCI, or 8 week after SCI) have reached the rostral graft-host 1994 transection (rat) microsurgical scissors and a glass aspiration pipette combined with a CL immediately border after 4 weeks, but a time- or 1 week prior to grafting course comparison showed a decrease after 8 weeks
Bilateral T9 Dorsal lamina of T9 removed, and the dura opened, a slit was E Intraspinal injections of NFG, Axonal tracing using CTB (1, Reduction in number of terminal Sayer et al., Hagg and complete DC cut perpendicular to the length of the spinal cord bilaterally BDNF, NT-3, combined in pairs 7 or 14 days after SCI) clubs at 1, 7 but not 14 days. Positive 2002 Oudega transection (rat) through the dorsal column including the corticospinal tracts or all together effects not significantly different with using microscissors individual or mixed neurotrophins
23 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader C3-4 SC Dura opened transversely at level C3-4 and dorsal hemisection E Intrathecal infusion of Gö6976 Axonal tracing using CTB (5 An increase in axons regenerating into Sivasankaran He hemisection was performed to a depth of 1.6 mm from the dorsal surface of (PKC inhibitor) weeks after SCI) and beyond the lesion (some fibers et al., 2004 (rat) the cord using a VibraKnife lesioning device made at beyond 6 mm) University of Louisville. The lesion was advanced to a level beyond the central canal to ensure that the entire bilateral dorsal funiculi were completely transected
Unilateral C2 Using the procedure of Richardson and Verge (1986), exposed C Unilateral L5 DRG injection of Axonal tracing using HRP (2 No difference in DRG neurons Han et al., Hoffman DC transection spinal cord by laminectomy at the C2 level and using dbcAMP and 7 days later months after SCI and regenerated axons between dbcAMP 2004 (rat) microscissors, symmetrically incised the dorsal columns to a transplantation the cut SN on the transplantation) and control groups, also no significant depth of approximately 1 mm. The sciatic nerve on the opposite side) + CL (1 week regeneration compared to CL uninjected side was cut immediately distal to the sciatic canal preconditioning unilateral sciatic and a 4-cm long segment removed for transplantation crush for 2x 30s)
High cervical The dorsal columns were incised bilaterally after high cervical C A segment from the right sciatic Axonal tracing using HRP (2-3 The long spinal axons of primary Richardson Issa DC transection laminectomy nerve was transplanted in the months after SCI) sensory neurons are 100 times more and Issa, 1984 (rat) midline to the injury. Spinal cord likely to regenerate into peripheral and peripheral conditioning lesion nerve grafts with a conditioning (cut and crush) was performed at transection of the peripheral nerve 1 multiple time points week prior to DC lesion
Individual Individual GFP+ axons emerging from the injected DRGs in C Viral gene delivery of rAAV- In-vivo confocal imaging of An increase in collateral and terminal Bareyre et al., Kerschen- cervical DC the dorsal funiculus on the cervical level using a hand-held 32- STAT3 into the DRGs of Thy1- fluorescently labeled DRG sprouting exclusively 2-4 days after 2011 steiner transections gauge hypodermic small-diameter needle were surgically re- GFP mice (10-12 days before neurons from Thy1-GFP mice SCI. Average axonal outgrowth was (mice) exposed and lesioned SCI), and application of ChABC (2-10 days after SCI) doubled by addition of ChABC
Bilateral T8 After a dorsal laminectomy at T8, dura was resected to expose C Anti-NG2 mAbs delivery into the Axonal tracing using CTB (4 Growth axons into and beyond the Tan et al., Levine complete DC the spinal cord. Using microscissors, the spinal cord was lesion site together with peripheral week after SCI) glial scar, with long-distance (up to 2006 transection (rat) transected bilaterally with the lesion extending ventrally to the nerve conditioning lesion (1 week 6.8 mm) with combination therapy. depth of the central canal (~1.5 mm) before SCI) Growth into lesion but not beyond with anti-NG2 alone
Superficial A small unilateral lesion was produced in Thy1-YFP mice by E Local administration of autophagy- Two-photon imaging of Thy1- An attenuation in axon retraction He et al., Luo unilateral C4-5 transecting only the superficial axons of the dorsal columns at inducing Tat-beclin1 immediately YFP mice (for 5h after SCI) distance 2016 transection the C4/C5 level with Vannas spring scissors after removing the after lesioning (mice) lamina
Bilateral T6 DC The dorsal columns were crushed at T6 with fine E Intrathecal delivery of NT-3, Transganglionic labeling with NT-3: An increase in fibre sprouting Bradbury et McMahon crush watchmakers’ forceps, the tips of which had been ground to BDNF and GDNF CTB for axonal tracing of the at lesion site and growth into and al., 1999 (rat) 0.3 mm in width. The tips of the forceps were held on either DC (4 weeks) beyond the crush site up to 4 mm. side of the dorsal columns, pushed 2 mm down into the cord GDNF promoted fibre growth then held tightly together for 10 s before raising them back out around caudal cavities but not into of the cord the epicenter. BDNF, no regeneration
24 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader T10-11 SC Following midthoracic laminectomy to expose the spinal cord E Nrg1 KO Functional: Electrophysiology, Ngr1 KO causes axonal conduction Bartus et al., McMahon contusion leaving the dura intact, animals received a moderate midline BMS and inclined beam failure and impaired functional 2016 and (mice) 50kdyne spinal contusion injury at spinal level T10/11 using walking test recovery in beam and BMS tests Bradbury an Infinite Horizons impactor (Precision Systems Instrumentation)
Bilateral C3 DC A bilateral lesion in the dorsal columns at spinal level C3 using E Transplantation with a TEG3 Axonal tracing using CTB (4 After 10 weeks there is an increase in Moreno- McMahon, crush watchmaker’s forceps (as previously described by Bradbury et OEG cell line or 10 weeks after SCI) axons inside the lesion, but not Flores et al., Wandosell (rat) al., 2002) Functional: Tape removal, beyond. Improved functional recovery 2016 and beam walking in tape and beam tests Bradbury
Bilateral C5 DC Using a needle, a small transverse hole was cut in the dura. I miRNA155 KO and peripheral Labeling sensory axons by Reduced axonal dieback (5 times Gaudet et al., Popovich crush “Guide” holes were created by inserting no. 5 forceps at the conditioning lesion (6 day injecting AAV2-GFP into both more CTB+ axons 0 - 200 µm caudal 2016 (mice) spinal cord midline, held at a width of 0.9 –1.0 mm with tips preconditioning bilateral sciatic sciatic nerves directly after SCI to the lesion) in wt and improved marked to a depth of 600um. Next, no. 55 forceps (labeled for ligation + transection distal to (perfused 32 days after SCI) axon regeneration (+11 µm, with depth) were inserted into the holes and closed for 3 x 10 s suture) max. 1.4 mm) in miRNA155 KO
C1-2 DC An insulated stainless steel electrode was inserted at C1-2 level, E Transplantation of SCs, Axonal tracing using HRP (2 Lesioned and intact axons adjacent to Li and Raisman electeocautery slightly to the side of the midline at a depth of 0.4-0.5 mm immediately after injury days or 2 weeks after SCI) the SC graft extended branches Raisman, (rat) and a constant anodal DC current of 10 µA was passed for 4-7 parallel to and inside the lesion/graft 1994 minutes site.
L3-4 bilateral The dorsal columns were lesioned close to the L3/4 segmental E Transplantation with different Axonal tracing using CTB and An increase of axons into cell Toft et al., Riddell complete DC border using a wire knife (David Kopf Instruments, Tujunga cocktails of olfactory cells, BDA (2-3 months after SCI) transplant, but not beyond. However, 2007 transection (rat) CA, USA). The sheathed knife was inserted through a slit in immediately after lesioning Functional: improved spinal cord function in the dura over the left dorsal root entrance zone, lowered to a Electrophysiological recordings sensory pathways assessed depth of 950 mm and then protruded to form an arc (~1.5 of CDPs and sensory-evoked electrophysiologically mm diameter) encompassing the dorsal columns potentials
Unilateral T8 The 6-mm spring microscissors was inserted underneath the C Combined immunomodulatory Anterograde axonal tracing A reduction in axonal dieback (1.7 ± Grosso et al., Steinmetz SC hemisection dura and, with the tips perpendicular to the spinal cord, a treatment, consisting of liposomal using Dex-TR (7 weeks after 0.49 µm vs. 0.55 ± 0.16 µm) of 2014 (rat) complete right-sided T8 lateral hemisection was created with clodronate and rolipram, along SCI) injured dorsal column axons, and the use of the microscissors with the CS-GAG-degrading Functional: Grid walk greatest improvement in locomotor enzyme, ChABC recovery at 6 weeks postinjury compared with controls and noncombined therapy
Bilateral T8-9 The lesion was performed utilizing specially-milled forceps E Injection with enriched p75+ Axonal tracing using CTB (2 Axonal regeneration into and Andrews and Stelzner DC crush (tips: 0.3 mm) placed at a depth of 1 mm from the dorsal (NGF receptor) OECs combined weeks after SCI) extending approx. 0.5 mm rostral to Stelzner, 2004 (rat) surface of the spinal cord crushing the dorsal funiculus with CL (7 days before SCI) lesion bilaterally at level T8-9, severing all DC axons (Bradbury et al., 1999)
25 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader T7 DC crush A jeweler's forceps (Dumont #7, Roboz, with 0.5 mm C Preconditioning lesion of the Axonal tracing using CTB (5 Triple therapy combining NgR1 Wang et al., Strittmatter lesion separation of the two tines) was inserted into the spinal cord at peripheral sciatic nerve, weeks after SCI) decoy, ChABC and preconditioning, 2012b (rat) T7 at a depth of 1.5 mm and held together in order to NgR1(310) ecto-Fc decoy protein allows ascending primary afferent complete the crush injury. The forceps was held together for treatment or ChABC treatment fibers to regenerate millimetres past 10 s before removal the spinal cord injury site
C4-5 complete A wire knife (model 120; David Kopf Instruments, Tujunga, E Intrathecal delivery of ROCK Axonal tracing using CTB (2 High dose animals exhibited Chan et al., Tetzlaff DC transection CA) was secured onto the stereotaxic frame and moved 0.9 inhibitor, Y27632, in the region of months after SCI) sprouting into grey matter and along 2005 (rat) mm lateral to midline. After a prepuncture of the dura mater the lesion (high and low dose) Functional: Footprint analysis, walls of lesion cavity. Footprint with a fine needle, the knife was lowered 1.1 mm into the controlled by osmotic minipumps horizontal ladder, food pellet analysis showed a faster normalization dorsal horn between the dorsal roots of C4 and C5. The wire reaching task, 10-point scale of base of support. Forepaw rotation knife blade (1.8 mm curvature length) was then extended into reaching task, heat withdrawal and number of footslips on horizontal the spinal cord through the prepuncture to a further depth of (Plantar IR apparatus) ladder and pellet reaching 0.5 mm (total 1.6 mm) then drawn up significantly improved compared to low dose animals. No allodynia detected in heat withdrawal
Bilateral T6 A partial laminectomy was performed at the T6 level. A small I Perineural infusion of JAK2 Transganglionic axonal tracing Significant attenuation of dorsal Qiu et al., Thompson complete DC incision was made in the dura, lignocaine applied to the inhibitor AG490 (blocks STAT3 using CTB (24 days after SCI) column axonal regeneration following 2005 crush opening. The dorsal column was crushed with fine phosphorylation) and CL (1 week peripheral nerve conditioning lesion (rat) watchmakers’ forceps. The tips of the forceps were held on preconditioning unilateral sciatic either side of the dorsal column, pushed 2 mm down into the transection) cord, held tightly together for 10 s
Bilateral T6 DC A small incision was made in the dura mater, and fine I IL-6 KO + CL (1 week Axonal tracing with CTB (4 IL-6 KO abolishes CL effect and fails Cafferty et al., Thompson crush (mice) watchmakers’ forceps were inserted into the spinal cord preconditioning unilateral sciatic weeks after SCI) to induce GAP43. 2mm axon growth 2004 (medial to the dorsal root entry zones) to a depth of 1 mm and axotomy) in CL held together for 10 sec before being removed. Crushing the dorsal columns with blunt forceps leaves the large midline dorsal blood vessel intact while entirely severing axons in the dorsal column projection at level T6
Bilateral C3 The dorsal funiculi were completely transected bilaterally at C MSCs expressing NT-3 Axonal tracing using CTB (4 Greater effects on central axon Blesch et al., Tuszynski complete DC C3 using a tungsten wire knife (Kopf Instruments, Tujunga, transplanted immediately after weeks after SCI) regeneration (maximum distance up 2012 transection (rat) CA) combined with dorsal tract compression to completely lesion combined with rostral to 2.2 mm) for CL applied prior or transect axons, as previously described (Lu et al., 2004; Taylor injection of LV-NT-3 and shortly after SCI combined with graft et al., 2006) peripheral conditioning lesion (7 d and viral delivery of NT-3 compared before, 1 d after or 7 d after SCI) to cAMP induced effects or cAMP injection 1 d before SCI
26 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader Bilateral C3 C3 dorsal columns were completely transected bilaterally using C Combinatorial treatment with Axonal tracing using CTB (13 Only animals that received triple Kadoya et al., Tuszynski complete DC a tungsten wire knife (Lu et al., 2004) MSC grafts secreting NT-3, weeks or 15 months after SCI) combinatorial treatment showed 2009 transection (rat) peripheral conditioning lesion and axonal bridging beyond the lesion for injection of LV-NT3 to the lesion both 13 weeks and 15 months site. Combination therapy was postinjury initiated either 6 weeks after SCI with a 6 week survival or 15 months after SCI (survival time not mentioned)
Bilateral C3 An acute dorsal column spinal cord lesion was first performed E Implants of MPC-NT-3-GFP (6 Axonal tracing using CTB (3 Robust axonal regeneration into Lu et al., 2007 Tuszynski complete DC at the C3 level, as described previously (Weidner et al., 2001). weeks after SCI) months after SCI) chronic lesion cavities expressing NT- transection (rat) A tungsten wire knife (Kopf Instruments, Tujunga, CA) was 3 stereotaxically positioned at the spinal dorsal midline, then moved 0.6 mm left laterally and lowered 1.1 mm from the dorsal spinal cord surface through a small slit in the dura. The knife blade was extruded 2.25 mm toward the midline, forming a 1.5 mm wide wire arc, subsequently raised 2 mm to ensure full bilateral transection
Bilateral C3 A Kopf microwire knife was used, as previously described (Lu E Implantation of +/-BDNF MSCs Axonal tracing using CTB (2 Native MSCs support modest growth Lu et al., 2005 Tuszynski complete DC et al., 2003; Weidner et al., 1999b). The wire knife was into epicenter of lesion (with an weeks, 1 or 3 months after of sensory neurons, BDNF expressing transection (rat) stereotaxically positioned at the spinal dorsal midline, then IRES-GFP reporter) SCI) MSCs resulted in significant increase moved 0.6 mm left laterally and lowered 1.1 mm from the Functional: Tape removal and of DC sensory axons. No functional dorsal spinal cord surface. The knife blade was extruded 2.25 rope-walking test recovery observed mm toward the midline, forming a 1.5-mm-wide wire arc. The arc was raised 2 mm and was simultaneously met by a blunt glass rod that added compression from above to ensure full transection of axons in the dorsal columns bilaterally
Bilateral C4 C4 dorsal columns were completely transected bilaterally using C Injection of cAMP into L4 DRGs Axonal tracing using CTB (1 An increase in number of axons that Lu et al., 2004 Tuszynski complete DC a Kopf wire knife device (David Kopf Instruments, Tujunga, 5 d before SCI. After SCI implants or 3 months after SCI) grew from 250 µm to 2000 µm transection CA) combined with tract external compression, as reported of BMSCs and injection of NT-3 Functional: Tape-removal test, beyond the lesion site. No functional (rat) previously (Weidner et al., 1999; Lu et al., 2003). into lesion cavity and an additional horizontal ladder locomotion recovery measured injection of NT-3 1 week test and a rope-walking test postinjury
Bilateral C3 C3 dorsal laminectomy, microwire knife was stereotaxically E Grafting of NSCs genetically Axonal tracing using CTB (2 Ascending sensory dorsal column Lu et al., 2003 Tuszynski complete DC positioned at the spinal cord midline. A small dural incision modified to express NT-3 weeks after SCI) and CGRP axons penetrated C17.2-NT-3 NSC transection (rat) was made, and the wire knife was lowered into the spinal cord IHC for nociceptive axon grafts more extensively than C17.2 to a depth of 1.1 mm ventral to the cord dorsal surface and 0.6 labeling NSC grafts (density measure) mm to the left of the midline. The tip of the wire knife was then extruded, forming a 1.5-mm-wide wire arc that was then raised to the dorsal surface of the cord
27 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader T7 SC The dorsal half of the spinal cord at level T7 was aspirated E Grafting of GDNF-secreting Axonal tracing using CTB (2 An increase in maximum distance Blesch and Tuszynski hemisection using a fine-tipped glass pulled device, as previously described syngenic fibroblasts weeks, 1 or 3 months after axon regeneration into grafts, however Tuszynski, (rat) (Grill et al., 1997; Blesch et al., 1999). The lesion was SCI). Neurofilament staining not beyond. Extensive growth into 2003 extended ventrally until all remnants of the corticospinal for general axonal growth, grafts (density measure) of CGRP, projection were verifiably aspirated under microscopic visual CGRP, subP and IB4 to label subP and IB4 labeled axons. No guidance. In the rostral-caudal plane, the lesion was 2–3 mm nociceptive axons functional recovery measured in BBB. in length Proprioceptive axons: 16 fold increase in regional fast blue and diamidino yellow propriospinal neurons with Functional: BBB projections into the lesion
Bilateral C4 A 30-gauge needle was inserted 1mm either side of the midline I AAV5 delivery of TFs to the left Axonal tracing using CTB (4 No observed increase in axon growth Fagoe et al., Verhaagen complete DC to 1.6 mm depth, enlarged with 27-gauge needle, cut with L4 and L5 DRG (c-jun, ATF3, weeks after SCI), fGFP 2015 and Mason transection microscissors Smad1, STAT3 or ATF3 alone) expressed with AAV (rat) Bilateral T8 Laminectomy at T8 subsequently a bilateral lesion (1.2 mm E Transplantation of genetically Axonal tracing using CTB (10 An increase of axon density 0-1.5mm Roet et al., Verhaagen complete DC deep) of the dorsal columns was made using a microknife (a modified fibroblasts expressing weeks after SCI) caudal to the lesion in SCARB2 2013 transection (rat) 25-gauge needle with a file-sharpened 1.2 mm tip bent 45°), as GFP, S100A9, SCARB2 or expressing group previously described (Hendriks et al., 2007) SERPINI1 (OECs)
Bilateral T8-9 The dorsal columns of T8 or T9 were transected with I Peripheral conditioning lesion Transganglionic axonal tracing Regrowth of DRG axons in Tenascin- Pasterkamp et Verhaagen complete DC microscissors, as deep as the central canal (some animals concomitant using CTB (1 or 2 months C and CSPG expressive scars, but no al., 2001 transection unilateral sciatic crush, others 1 after SCI) penetration from DC fibers in (rat) week preconditioning unilateral Sema3A and CNS myelin positive sciatic transection) scars
Bilateral T10 DC lesion was performed by a hemi-laminectomy at T10, I Transgenic mouse line expressing Axonal tracing using HRP (4 The regenerative effect of the CL is Seijffers et al., Woolf complete DC transecting the dorsal column on both sides with ophthalmic constitutively active ATF3 weeks after SCI) not replicated in constitutively active 2007 transection microscissors combined with CL (4 day ATF3 animals. CL animals ~0.35 mm (mice) preconditioning unilateral sciatic axon growth from centre of lesion, 15 s crush) control -0.15 mm
Bilateral T6-7 Transection was performed using a tungsten steel I Peripheral CL (unilateral sciatic Neuronal tracing with B-HRP An increase in outgrowth of neurites Neumann and Woolf complete DC microscissors. DC was cut from one DREZ to the other down ligation + transection distal to (8 weeks after SCI) from the transected DC fibers ~2.5 Woolf, 1999 transection to the central canal suture) at different time points mm from lesion site, considerable (rat) sprouting
T11-12 lateral After laminectomy at T12, stainless clamps of stereotaxic E Postinjury intraperitoneal Two-photon imaging of YFP- An attenuation of progressive damage Tang et al., Zhang hemisection apparatus used to immobilize the spinal column, then we used administration of MP in YFP-H H line and Thy1-GcaMP mice of axons, increased blood flow, and 2015 (mice) microsurgical forceps and microscissors to tear the dura of the line and Thy1-GcaMP mice Functional: BMS reduced calcium influx postinjury and spinal cord segment. A sharp scalpel of 150 µm width was functional recovery over 90 days used to cut through to the ventral cord, then transected the whole left spinal cord to the lateral side. Glass window implanted above lesion
28 Type of lesion Technical approach Experimental intervention Histological & functional Result Reference Lab/Group (model) I/E/C Approach assessment leader Bilateral C1 Lesions were made with a Scouten wire-knife 1.25 mm lateral C The lesion cavity was filled with Axonal tracing using CTB and Increased numbers of synaptic Hollis et al., Zou complete DC to midline extending in an arc 2.5 mm wide and 1 mm deep ±200.000 BMSCs, either naïve or fluorogold (16 weeks after connections between proprioceptive 2015 transection SFRP2-positive (blocks repulsive SCI), staining of presynaptic sensory axons and DC neurons (rat) WNT signaling). Conditioning Vglut1 accompanied by improved effect was initiated by injecting Functional: Beam crossing, proprioceptive function in CL and EtBr into the sciatic nerve 1 week rotarod, Von Frey test, CL/SFRP+. Newly recovered prior to lesion Hargreaves test proprioceptive function is lost after silencing or severing the DC caudal to injury
Bilateral C5 For the dorsal column lesion, a C5 laminectomy was I Targeted pharmacological Axonal tracing using DexTR An increase in fibres presence in the Finelli et al., Zou complete DC performed and ascending sensory fibers were transected using modulation of histone deacetylases (2 weeks after SCI) center of the glial scar (MS-275 more 2013 transection iris microscissors (Fine Science Tools) to a depth of 0.8 mm activity, TSA (Class I/II HDACi) robust than TSA) (mice) or MS-275 treatment (HDAC1- specific inhibitor)
Bilateral C4 C4 laminectomy, dorsal columns lesioned with two passes of a E Transplantation with BMSCs Axonal tracing using CTB (1 Blocking CL induced Wnt signaling Hollis and Zou complete DC Scouten wire-knife (David Kopf Instruments) at 1.2 mm expressing either Wnt4, WIF1 or month after SCI) with WIF1 or SFRP2 expressing Zou, 2012 transection (rat) lateral to the midline and at a depth of 1 mm. The intact dura SFRP2, immediately after injury, 1 grafts resulted in enhanced central was pressed against the wire-knife to ensure complete week after sciatic nerve crush CL branch regeneration while Wnt4 transection of ascending dorsal column sensory axons expressing grafts caused dramatic long range retraction Bilateral T8 The lamina of T8 spinal segment was exposed and the dorsal I Intrathecal AAV delivery of Axonal tracing using DexTR AAV-BMP4 delivery resulted in less Parikh et al., Zou complete DC columns transected bilaterally using iris microscissors (Fine Smad1-dependent BMP (AAV- (4 weeks after SCI) retraction of long-projection sensory 2011 transection Science Tools), with the depth reaching ~0.8 mm BMP4) 2 weeks prior to DC injury Functional: Von Frey test fibers compared to vehicle & GFP (mice) indicates mechanical allodynia controls in L5 sciatic nerve ligation animals only, from 7-14 days
Key: Anatomy: DC = dorsal column, C = cervical, T = thoracic, L = lumbar, DRG = dorsal root ganglion, DREZ = dorsal root entry zone, CSF = cerebrospinal fluid. Immunohistochemistral: CTB = cholera toxin B, HRP = horseradish peroxidase, HRP-B = choleragenoid HRP, BDA = biotinylated dextran amine, DexTR = Texas red-conjugated dextran, GFP = green fluorescent protein, eGFP = enhanced GFP, fGFP = farnesylated GFP, GCaMP = GFP calmodulin peptide, CGRP = calcitonin gene-related peptide, IHC = immunohistochemistry. Factors: NT = neurotrophin, GDNF = glial cell-derived neurotrophic factor, CNTF = ciliary neurotrophic factor, BDNF = brain derived neurotrophic factor, NGF = nerve growth factor, bFGF = basic fibroblast growth factor, BMP-4 = bone morphogenetic protein 4, STAT3 = signal transducer and activator of transcription 3, ATF3 = activating transcription factor 3, Smad1 = mothers against decapentaplegic homolog 1, c-jun = transcription factor AP-1, cAMP = cyclic adenosine monophosphate, dbcAMP = dibutyryl cAMP, PKC = protein kinase C, CREB = cAMP responsive element binding protein, PST = polysialyltransferase, MP = methylprednisolone, epoB = epothilone B, PGB = pregabalin, tPA = serine protease tissue-type plasminogen activator, PCAF = p300/CBP-associated factor, ChABC = chondroitinase ABC, SLPI = secretory leukocyte protease inhibitor, IL-6 = interleukin 6, FBS = fetal bovine serum, ROCK = Rho-associated protein kinase, UbC = ubiquitin C, NG2 = neural/glial antigen 2, Nrg1 = neuregulin 1, JAK2 = janus kinase 2, Sema3A = semaphorin 3A, SFRP2 = secreted frizzled related protein 2, Vglut1 = vesicular glutamate transporter 1, EtBr = ethidium bromide, HDAC = histone deacetylase, TSA = trichostatin-A, MS-275 = entinostat (commercial name), WIF1 = wnt inhibitory factor 1, C1q = complement 1q complex. Donor cell types: SC = Schwann cell, OEC = olfactory ensheathing cell, OEG = olfactory ensheathing glia, (B)MSC = (bone) marrow stromal cells, GRP = glial-restricted precursors, NRP = neuronal-restricted precursors. Functional tests: BBB = Basso Beattie Bresnahan, BMS = Basso mouse scale, EMG = electromyography. Viral: LV = lentivirus, AAV = adeno-associated virus, VP = viral protein.
29 Table 2: Overview of the studies that conducted experimental DC lesions paradigms with a description of their technicial approach, the histological and functional assessment of axon regeneration and functional recovery and their main results, without any experimental intervention.
Type of lesion Technical approach Histological assessment Functional assessment Result Reference Lab/Group (model) leader DC: Bilateral DC: The cord rostral to each rat’s C4 dorsal roots entry DC: Axonal tracing with CTB DC: Sticker attention, An increase in reaction time to Onifer et Magnuson C4 complete zones were cut dorsal–ventral transversely to a depth of (1 week after SCI) electrophysiological assessment after sticker attention in DC, DF, al., 2005 DC transection 1.1 mm from the lacerated dorsal spinous vein with a behavioral tests (1 week after SCI) DHx but not in DLF lesioned (rat) custom-made 0.12-mm-thick and 1.6-mm-wide animals after 1 week diamond-shaped (the tip angle was 90º) piece of a single-edge razor blade attached to the Vibraknifek, a Increased reaction time is modified VIBRATOMER Series 1000 dampened after 3-4 weeks in DHx lesioned animals that were DF: Bilateral DF: The left and right dorsal funiculi of seven rats DF: Axonal tracing with CTB DF: Sticker attention, assessed weekly. However, C4 complete randomly assigned to Group III were completely (1 week after SCI) electrophysiological assessment after significant increase again when DF transection transected by cutting the cord rostral to the C4 dorsal behavioral tests (1 week after SCI) only measured at 4 weeks (rat) roots entry zones dorsal–ventral transversely to a depth postinjury which indicated that of 1.5 mm from the lacerated dorsal spinous vein with weekly testing promoted recovery the 1.6-mm wide, diamond-shaped piece of razor blade through learning
DLF: Bilateral DLF: The left and right dorsolateral funiculi, but not DLF: Axonal tracing with DLF: Sticker attention, C4 complete the left and right dorsal funiculi, rostral to the C4 CTB (1 week after SCI) electrophysiological assessment after dorsolateral dorsal roots entry zones of eight rats randomly assigned behavioral tests (1 week after SCI) funiculi to Group IV were cut dorsal–ventral transversely to a transection (rat) depth of 1.5 mm. The starting position for this lesion was where a 0.8-mm-wide piece of razor blade contacted the spinal cord’s dorsal surface with one edge of the blade positioned over the lateral edge of the spinal cord. In this case, the lateral excursion of the razor blade produced a lesion that at its maximum depth extended medially 1 mm from the cord’s lateral edge DHx: C4 dorsal DHx: Axonal tracing with DHx: (a) Sticker attention, hemisection DHx: To completely sever the left and right dorsal CTB (1 week after SCI) electrophysiological assessment after (rat) funiculi plus the left and right dorsolateral funiculi of behavioral tests (1 week after SCI), (b) 22 rats randomly assigned to Groups V, VII, and IX, weekly sticker attention and grid the dorsal, but not ventral, half of the cord rostral to walking with electrophysiological the C4 dorsal root entry zones was hemisected dorsal– assessment (4 weeks after SCI), (c) ventral transversely to a depth of 1.5 mm from the sticker attention and lacerated dorsal spinous vein with a 3.8-mm-wide piece electrophysiological assessment (4 of razor blade weeks after SCI)
30 Type of lesion Technical approach Histological assessment Functional assessment Result Reference Lab/Group (model) leader Bilateral C2 or Using a modified sterile 25 gauge hypodermic needle, Overground locomotion and C2 DCL resulted in persistent Kanagal and Muir T7-8 complete lesions were made bilaterally to either the cervical (C2) horizontal ladder (2 and 6 weeks after errors in both functional tests. In Muir, 2008 DC transection or mid-thoracic (T7-8) dorsal funiculus (dorsal SCI) contrast, T7-8 DCL did not (rat) columns with dorsal corticospinal tract) affect over ground locomotion and only caused minor errors in horizontal ladder at 2w postinjury, with recovery to pre- surgical levels 6 w after injury T8 SC The spinal column was stabilized using a spinal clamp AAV8-UbC-GFP was Soderblom Tsoulfas contusion and the exposed T8 cord placed below the impacter of delivered into the CSF et al., 2015 (rat) an Infinite Horizon Impactor device (Precision Systems through the cisterna magna and Instrumentation, 200 kdynes) (10 days before SCI). 8-9 weeks after SCI 3d reconstruction of confocal images from cleared DRG at C3, T9, and L5 levels labeled with AAV8-UbC-GFP
Bilateral C4 To minimize compression damage of the spinal cord Axonal tracing using CTB, Adhesive tape removal test, horizontal Generally function recovered to Fagoe et al., Verhaagen and T7 we first inserted a 30G needle at 1mm (at C4) or 0.6 gracile nucleus assessed for ladder, rope crossing test, CatWalk gait sham levels within 2-6 weeks and 2016 Mason complete DC mm (at T7) lateral to the midline on either side to a spared axons (8 weeks after analysis and the inclined rolling ladder. C4 lesions were more severe than transection depth of 1.6mm (at C4) or 1.4mm (at T7). The SCI) Hindlimbs assessed only T7 (rat) resulting hole was then enlarged by inserting a 27G needle to the same depth. Finally the tips of a pair of micro-scissors were inserted in the same holes to the same depth and then closed
Unilateral C1 A sagittal incision was made in the nape of the neck, Reaching task, force measurement, Normal performance levels on all Ballermann Whishaw DC transection and the C1 and C2 vertebrae were exposed through haptic discrimination, vertical paw tests with the exception of et al., 2001 (rat) blunt dissection. The medial part of the dorsal arch of placing, adhesive dot removal deficits in haptic discrimination C1 was removed with a drill, and the dorsal column when feeling for a food/ non- was cut using a sharp No. 11 razor blade. The food item. transections were made on the ipsilateral side to the preferred paw for reaching
Unilateral C2 A sagittal incision was made at the nape of the neck, Reaching task and rotary limb Reaching success completely McKenna Whishaw DC transection blunt dissection revealed the dorsal surface of the first movement analysis recovered within a few days of and (rat) and second cervical vertebrae. The mediocaudal part of dorsal column lesion. Whishaw the C1 vertebra was removed with rongeurs, so that the Compensation was achieved with 1999 dorsal column tract on one side of the spinal cord was whole-body and alternate limb made visible and could be incised with a sharp No. 11 movements (which were scalpel blade irreversibly impaired)
31
Chapter 5
Conclusion
Over the last decades, our knowledge on SCI extensively expanded and several models, the DC lesion model amongst others, for injury to the spinal cord has provided a solid fundament for future research. Specifically, the DC lesion model is characterized by its uniqueness in terms of the anatomical features of the DRG neurons and the corresponding regenerative capacity of both the peripheral and central DRG axons. For this reason, the DC lesion model is exceptionally fit for neuroregenerative research and already a lot has been learned from previous studies on positive and negative regulators in this model.
However, there is delicate balance matching a specific DC lesion model to the correct assessment of axon regeneration and functional recovery in SCI research. Generally, the functional deficits after a spinal cord lesion reflect the severity of the injury and the assessment of sensory function usually reflect the extent of recovery upon injury. The terms in which improvement of central DRG axon regeneration and functional recovery of sensorimotor and proprioceptive tasks has been established, is heavily subjected to the huge amount of variation in conducting DC lesions. There is too much variation in surgical procedures, histology and functional testing, leading to ambiguity in their interpretation in this model. This support the notion of standardization of the experimental model for DC injury. Notably, re-growth of injured DC axons is in most studies exclusively assessed using histological tracers, without connecting the enhanced axon regeneration to recovery of sensory function. Uncoupled improvements in either histologically assessed axonal outgrowth and functionally assessed sensory function could be caused by a wrong methodological setup. The functional analysis of regeneration is unavoidable and therefore emerging imaging techniques, such as magnetic resonance imaging or 3D imaging of cleared spinal cords, and electrophysiological approaches are extremely meaningful for future research and could help with histologically established pro-regenerative results of experimental interventions. These new emerging techniques could play a great role in providing insight in alternative regeneration pathways that are overlooked by histology and on supposedly compensatory adaptive plasticity of local circuitry and spared tracts that could explain improved sensory function.
In conclusion, many therapeutic strategies have been advanced using various DC lesion models matching their scientific question. Testing of new intrinsic experimental interventions is supposedly best to combine with a DC transection model. When the intervention can be shown to establish injured axons to regenerate, sprout and form new circuits, they should probably be combined with an extrinsic treatment and tested in a more clinically relevant crush or contusion model for DC injury.
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