Concepts and methods for the study of axonal regeneration in the CNS

Abstract

Progress in the field of axonal regeneration research has been like the process of axonal growth itself: there is steady progress toward reaching the target, but there are episodes of mistargeting, misguidance along false routes, and connections that must later be withdrawn. This primer will address issues in the study of axonal growth after central nervous system injury in an attempt to provide guidance toward the goal of progress in the field. We address definitions of axonal growth, sprouting and regeneration after injury, and the research tools to assess growth.

Figure 1. Regeneration and Sprouting

The left side of the panel illustrates several examples of types of regeneration. (A) Intact axon. (B) Transected or crushed axon. (C) Canonical regenerating axon: new growth occurs from tip of transected axon leading to reinnervation of its normal target. (D) Regenerating axon, wherein new growth arises not from tip of transected axon, but from region of axon close to injury site. In some literature, this is referred to as “regenerative sprouting”, a term we avoid because it can generate confusion. (E) Another example of a regenerating axon, wherein new growth arises from a transected axon, but from a region of the axon that is remote from the injury site. This type of growth has been described to arise from CST axons in the cervical spine cord after thoracic level injuries (Bareyre et al., 2004). This has also been referred to as “regenerative sprouting.” (F) An example of canonical sprouting: here damage to one pathway induces compensatory growth of new connections from nearby undamaged axons.

Figure 2. Anterograde or Retrograde Labeling to Identify New Axonal Growth

An example of anterograde axonal labeling to identify new axonal growth and putative regeneration is shown in panels A–D (from (Lu P, 2012)). Double retrograde labeling as a means of demonstrating new axonal growth and putative regeneration is shown in panel E (from Wolfram Tetzlaff, Univ. British Columbia). (A) After T3 complete transection, darkly labeled reticulospinal axons approach the lesion site from the left (rostral) direction in these 35μm-thick horizontal sections of the spinal cord. A graft (g) of bone marrow stromal cells is present in the lesion site. The border between the host spinal cord and the graft in the lesion site is indicated by black dashed lines. Darkly labeled axons are seen penetrating the graft (B), and some of these axons have grown to the caudal aspect of the graft occupying the lesion site, and beyond this point into the host spinal cord below the lesion site (C–D). The inset indicates one plane of the complete transection site by GFAP immunolabeling. Green indicates expression of Brain Derived Neurotrophic Factor (BDNF) and a reporter gene expressed by a lentiviral vector injected into the spinal cord below the lesion to attract growing axons; IS indicates vector injections sites. (B) In the graft adjacent to the rostral host/graft interface, numerous host axons have penetrated the graft. (C) At the distal graft (g) / host (h) interface, darkly labeled axons cross from the graft into the spinal cord below. Dashed lines indicate the border between graft and host. Note the irregular morphology and trajectories of axons growing beyond the lesion site. Axons are not in tightly fasciculated bundles typical of intact axons, but instead exhibit highly varied and branched morphologies, are dispersed from one another, and turn in various directions. (D) 1.5mm caudal to the lesion site, darkly labeled axons remain detectable and continue to exhibit varying trajectories. This continues 4mm caudal to the lesion. (E) Red nucleus in the brainstem of the rat. Rats were injected with the retrograde tracer Fast Blue at cervical segment 8 to back label intact rubrospinal neurons. The spinal cord was lesioned at the 4^th cervical segment eight days later. After a delay, injured rats were treated with a sciatic nerve graft in the lesion site and BDNF infusions. Two months later, a second retrograde tracer was placed at the free end of the sciatic nerve segment in the lesion site, and the brainstem was subsequently examined. Some cells in the red nucleus were single labeled for Fast Blue (arrowheads), and some were double labeled for Fast Blue and BDA (visualized with the fluorophore Avidic-CY3). Double-labeled cells represented neurons that grew axons into the peripheral nerve graft; single labeled cells represented neurons that did not grow axons into the lesion site. Scale bar A, 1mm; A inset, 0.8mm; B, 70μm; C, 150μm; D, 70μm; E, 3μm.

Figure 3. Spinal Cord Dorsal Column Sensory Regeneration Model

The dorsal columns of the spinal cord contain axons that ascend from the lumbar region to the nucleus gracilis in the medulla. These axons can be traced as they ascend the spinal cord by injections of the transganglionic tracer Cholera Toxin B subunit (CTB) into the sciatic nerve. (A) In animals that undergo C3 dorsal column transection lesions, CTB labeled dorsal column sensory axons approach the lesion site, but few penetrate a graft of bone marrow stromal cells placed in the lesion site; grafted cells express the reporter gene GFP (from (Lu et al., 2004). Sagittal, 35μm-thick section. (B) A higher magnification view at the light level shows the approach of axons to the lesion site (upper right), and rare axons in the graft (graft outlined within black lines). Arrow indicates a single axon that reaches the rostral graft/host interface, but does not regenerate beyond the lesion site. (C) Following combinatorial therapy with a conditioning lesion of the sciatic nerve, a marrow stromal cell graft in the lesion cavity, and lentiviral NT-3 growth factor delivery rostral to the lesion, numerous axons penetrate the graft and regenerate beyond it. Rostral aspect of graft is outlined by dashed lines. Regenerating axons within and beyond the graft are indicated by arrowheads. (D) At the rostral host-graft interface, numerous CTB-labeled axons regenerate out of the graft and into host white matter beyond the lesion. Axons exhibit irregular trajectories, make abrupt turns, and are generally dispersed, features typical of regenerating axons. (E) At a distance of 2mm rostral to the lesion site, regenerating host axons are present in adult white matter. Arrowheads indicate individual axons. From Alto et al., (2007) and Blesch et al., (2012). (F) Lesion completeness in this model can be confirmed by examination of the medulla. In intact animals, dense reaction product is evident in nucleus gracilis after injection of CTB into the sciatic nerve. Gr, gracilis; Sol, soleus. Transverse 35μm-thick section. (G) In contrast, the nucleus gracilis is devoid of CTB labeling in animals that have undergone complete C4 dorsal column lesions. Scale bar = A, 250μm; B, 500μm; C, 1mm; D-E, 20μm; F-G, 200μm.

Figure 4. The Corticospinal Tract (CST)

(A, B) Corticospinal axons in a mouse are traced here by making placing four injections of mini-ruby BDA into the right sensorimotor cortex. CST axons from the right hemisphere are labeled. In mice, CST axons descend primarily in two tracts: the dorsal CST, in the ventral part of the dorsal column (dCST), and the dorsolateral CST (dlCST) in the dorsal part of the lateral column. Note the absence of labeled axons in the ventral column on the right side; in rats, some axons are present in this region. Note also the presence of a few labeled axons in the dorsal column on the right in the same location as the dCST. This illustrates the fact that there are a small number of axons that descend in the spinal cord ipsilateral to the cortex of origin. In this case, labeled CST axon arbors are found mainly in the ventral horn gray matter. The distribution of axonal arbors depends on whether the injections target mainly the sensory vs. motor divisions of the sensorimotor cortex. Injections that mainly target the primary motor cortex preferentially label CST axon arbors in the ventral horn whereas injections that mainly target the sensory cortex preferentially label CST axon arbors in the dorsal horn. (C) Schematic illustration of partial transection lesions that are commonly used for assessing CST regeneration in mice and rats. The red region denotes a dorsal hemisection; the blue region indicates a “T” lesion, which is intended to include the ventral CST in rats (modified from Zheng et al., 2006). (D, E) Corticospinal axons in a rat are traced here by making six injections of mini-ruby BDA into the right sensorimotor cortex. Labels are as in A, B. In this case, labeled axonal arbors are found mainly in the dorsal horn gray matter. (F) higher magnification view of BDA-labeled axons in the ventral column (vCST). Scale bars = 250μm. (G, K) Sprouting of CST axons below a hemisection injury in rhesus monkeys. (G) The schematic on the left illustrates the organization of the CST in rhesus monkeys. Descending CST axons from the left motor cortex are shown. The main component of CST axons descend in the lateral column, as in humans. About 87% descend on the side contralateral to the cortex of origin; 11% descend through the lateral column on the ipsilateral side; the ventral CST contains about 2% of the total number of labeled axons. The schematic on the right illustrates the lesion model, a hemisection at C7, and the CST axons that would survive such lesions. The box indicates the CST arbors in the gray matter that arise from axons decussating across the spinal cord midline. (H) Density of BDA-labeled CST axon arbors in the gray matter in intact monkeys (which includes labeled axons from the main tract and the crossing axons); (I) arbors of surviving crossed CST axons at early time points after a C7 hemisection; (J) arbors of crossed CST axons traced 8 months after a hemisection injury, when extensive compensatory sprouting has occurred. (K) Quantitative assessment of the density of crossed CST axons in the gray matter in intact monkeys, at short intervals after the injury, and at long post-injury intervals. By eight months post-lesion, there is a substantial reconstitution of corticospinal axons below the lesion site, due to sprouting of spared contralateral axons. Panels G-K are from Rosenzweig et al., 2010. Scale bar in “I” = 100μm, and applies to H–J.

Figure 5. CST Tract Reconstruction

CST axons were anterogradely traced with BDA in a PTEN-deleted mouse that survived for a total of 12 weeks following a complete spinal cord crush at T8. The image illustrates BDA-labeled axons in 50 μm thick serial sagittal sections. Labeled axons in each image were traced in Adobe® Photoshop®, and the tracings were stacked and superimposed onto a light micrograph of one section containing the central canal. Axon segments are rainbow color-coded according to depth of each of the 8 serial tissue sections, thus color-coding relative depth in 50 μm increments through 400 μm of tissue. Order of colors is red, orange, yellow, yellow green, green, cyan, blue, purple. This image is from the case described in Liu et al., 2010 in which sections were embedded and sectioned for electron microscopy in order to locate BDA labeled synapses. (Image courtesy of Rafer Willenberg, UC Irvine.)

Figure 6. Raphe-Spinal Pathways

(A) Retrogradely labeled neurons in the midline raphe and reticular formation after injections of true blue into the spinal cord of a mouse. Neurons in the raphe nucleus give rise to serotonergic (5HT) axons that project to the spinal cord. (B) Immunofluorescence for 5HT in the thoracic spinal cord. CC = central canal. (C) Immunolabeling for 5HT after a “complete crush” injury in mice. The lesion site is indicated. Scale bars = 250μm.

Figure 7. Brainstem Motor Pathways: Rubrospinal, Reticulospinal and Vestibulospinal tracts

A–C: Retrogradely labeled neurons in the brainstem after injections of true blue into the spinal cord of a mouse. (A) red nucleus; (B) vestibular nucleus; (C) locus ceruleus and Barrington’s nucleus. D–F illustrate BDA labeled axons after a large injection of BDA into the brainstem of a mouse. RuST, rubrospinal axons; RST, reticulospinal axons; VST, vestibulospinal axons. Note collaterals extending from the RuST into the gray matter in E. Collaterals in F are likely from the RST or VST. Scale bars = 250μm.

Figure 8. Three Dimensional Imaging of the Unsectioned Spinal Cord

Using tetrahydrofuran-based methods, the unsectioned adult rat spinal cord can be “cleared,” supporting visualization of the course of individual, fluorescently labeled axons through a spinal cord lesion site (Erturk et al., 2012). This supports tracing the origin and course of individual, lesioned axons. (A) The whole-mounted spinal cord in a 3D representation, showing axons labeled in transgenic M mice (Feng et al., 2000) expressing GFP in sparse neuronal populations (Erturk et al., 2012). (B) Reconstruction of a plane of section from the same spinal cord demonstrating GFP-labeled sensory axons (arrows) approaching and growing within a lesion site (lesion margins indicated by dashed lines). Rats underwent peripheral “conditioning” lesions of the sciatic nerve to enhance regeneration. Caudal is to the right, rostral to the left; the direction of axonal regeneration is right to left. (Courtesy of A Erturk and F Bradke.)

Figure 9. Genetic Tract Labeling

In vivo gene delivery vectors with dual promoters can identify specific neurons in vivo that express a candidate regeneration gene of interest, and can label the axonal projections of these neurons exclusively. In this case, AAV2 vectors expressing a candidate regeneration gene and the copGFP gene have been injected into the rat motor cortex. The axonal projections of these neurons in the spinal cord exhibit copGFP labeling (Low et al., 2010). (A) Corticospinal axons expressing copGFP are labeled with a light-level antibody as they approach a C3 dorsal column lesion site; bone marrow stromal cells have been grafted (g) into the lesion. (B) In a different field, individual axons that have incorporated the copGFP reporter are shown with fine morphological detail.