Matt S. Ramer

Functional regeneration of sensory axons into the adult spinal cord

The arrest of dorsal root axonal regeneration at the transitional zone between the peripheral and central nervous system has been repeatedly described since the early twentieth century. Here we show that, with trophic support to damaged sensory axons, this regenerative barrier is surmountable. In adult rats with injured dorsal roots, treatment with nerve growth factor (NGF), neurotrophin-3 (NT3) and glial-cell-line-derived neurotrophic factor (GDNF), but not brain-derived neurotrophic factor (BDNF), resulted in selective regrowth of damaged axons across the dorsal root entry zone and into the spinal cord. Dorsal horn neurons were found to be synaptically driven by peripheral nerve stimulation in rats treated with NGF, NT3 and GDNF, demonstrating functional reconnection. In behavioural studies, rats treated with NGF and GDNF recovered sensitivity to noxious heat and pressure. The observed effects of neurotrophic factors corresponded to their known actions on distinct subpopulations of sensory neurons. Neurotrophic factor treatment may thus serve as a viable treatment in promoting recovery from root avulsion injuries.

Minocycline Treatment Reduces Delayed Oligodendrocyte Death, Attenuates Axonal Dieback, and Improves Functional Outcome after Spinal Cord Injury

Minocycline has been demonstrated to be neuroprotective after spinal cord injury (SCI). However, the cellular consequences of minocycline treatment on the secondary injury response are poorly understood. We examined the ability of minocycline to reduce oligodendrocyte apoptosis, microglial/macrophage activation, corticospinal tract (CST) dieback, and lesion size and to improve functional outcome after SCI. Adult rats were subjected to a C7-C8 dorsal column transection, and the presence of apoptotic oligodendrocytes was assessed within the ascending sensory tract (AST) and descending CST in segments (3-7 mm) both proximal and distal to the injury site. Surprisingly, the numbers of dying oligodendrocytes in the proximal and distal segments were comparable, suggesting more than the lack of axon-cell body contiguity played a role in their demise. Minocycline or vehicle control was injected into the intraperitoneal cavity 30 min and 8 hr after SCI and thereafter twice daily for 2 d. We report a reduction of apoptotic oligodendrocytes and microglia within both proximal and distal segments of the AST after minocycline treatment, using immunostaining for active caspase-3 and Hoechst 33258 staining in combination with cell-specific markers. Activated microglial/macrophage density was reduced remote to the lesion as well as at the lesion site. Both CST dieback and lesion size were diminished after minocycline treatment. Footprint analysis revealed improved functional outcome after minocycline treatment. Thus, minocycline ameliorates multiple secondary events after SCI, rendering this clinically used drug an attractive candidate for SCI treatment trials.

Wallerian degeneration: Gaining perspective on inflammatory events after peripheral nerve injury

In this review, we first provide a brief historical perspective, discussing how peripheral nerve injury (PNI) may have caused World War I. We then consider the initiation, progression, and resolution of the cellular inflammatory response after PNI, before comparing the PNI inflammatory response with that induced by spinal cord injury (SCI).In contrast with central nervous system (CNS) axons, those in the periphery have the remarkable ability to regenerate after injury. Nevertheless, peripheral nervous system (PNS) axon regrowth is hampered by nerve gaps created by injury. In addition, the growth-supportive milieu of PNS axons is not sustained over time, precluding long-distance regeneration. Therefore, studying PNI could be instructive for both improving PNS regeneration and recovery after CNS injury. In addition to requiring a robust regenerative response from the injured neuron itself, successful axon regeneration is dependent on the coordinated efforts of non-neuronal cells which release extracellular matrix molecules, cytokines, and growth factors that support axon regrowth. The inflammatory response is initiated by axonal disintegration in the distal nerve stump: this causes blood-nerve barrier permeabilization and activates nearby Schwann cells and resident macrophages via receptors sensitive to tissue damage. Denervated Schwann cells respond to injury by shedding myelin, proliferating, phagocytosing debris, and releasing cytokines that recruit blood-borne monocytes/macrophages. Macrophages take over the bulk of phagocytosis within days of PNI, before exiting the nerve by the circulation once remyelination has occurred. The efficacy of the PNS inflammatory response (although transient) stands in stark contrast with that of the CNS, where the response of nearby cells is associated with inhibitory scar formation, quiescence, and degeneration/apoptosis. Rather than efficiently removing debris before resolving the inflammatory response as in other tissues, macrophages infiltrating the CNS exacerbate cell death and damage by releasing toxic pro-inflammatory mediators over an extended period of time. Future research will help determine how to manipulate PNS and CNS inflammatory responses in order to improve tissue repair and functional recovery.

Nerve growth factor induces P2X3 expression in sensory neurons

Glial cell line‐derived neurotrophic factor (GDNF) and nerve growth factor (NGF) are neuroprotective for subpopulations of sensory neurons and thus are candidates for pain treatment. However, delivering these factors to damaged neurons will invariably result in undamaged systems also being treated, with possible consequences for sensory processing. In sensory neurons the purinergic receptor P2X3 is found predominantly in GDNF‐sensitive nociceptors. ATP signalling via the P2X3 receptor may contribute to pathological pain, suggesting an important role for this receptor in regulating nociceptive function. We therefore investigated the effects of intrathecal GDNF or NGF on P2X3 expression in adult rat spinal cord and dorsal root ganglia (DRG). In control spinal cords, P2X3 expression was restricted to a narrow band of primary afferent terminals within inner lamina II (IIi). Glial cell line‐derived neurotrophic factor treatment increased P2X3 immunoreactivity within lamina IIi but not elsewhere in the cord. Nerve growth factor treatment, however, induced novel P2X3 expression, with intense immunoreactivity in axons projecting to lamina I and outer lamina II and to the ventro‐medial afferent bundle beneath the central canal. In the normal DRG, we found a greater proportion of P2X3‐positive neurons at cervical levels, many of which were large‐diameter and calcitonin gene‐related peptide‐positive. In both cervical and lumbar DRG, the number of P2X3‐positive cells increased following GDNF or NGF treatment. De novo expression of P2X3 in NGF‐sensitive nociceptors may contribute to chronic inflammatory pain.

Setting the stage for functional repair of spinal cord injuries: a cast of thousands

Here we review mechanisms and molecules that necessitate protection and oppose axonal growth in the injured spinal cord, representing not only a cast of villains but also a company of therapeutic targets, many of which have yet to be fully exploited. We next discuss recent progress in the fields of bridging, overcoming conduction block and rehabilitation after spinal cord injury (SCI), where several treatments in each category have entered the spotlight, and some are being tested clinically. Finally, studies that combine treatments targeting different aspects of SCI are reviewed. Although experiments applying some treatments in combination have been completed, auditions for each part in the much-sought combination therapy are ongoing, and performers must demonstrate robust anatomical regeneration and/or significant return of function in animal models before being considered for a lead role.

Neurotrophin-3-mediated regeneration and recovery of proprioception following dorsal rhizotomy.

Injured dorsal root axons fail to regenerate into the adult spinal cord, leading to permanent sensory loss. We investigated the ability of intrathecal neurotrophin-3 (NT3) to promote axonal regeneration across the dorsal root entry zone (DREZ) and functional recovery in adult rats. Quantitative electron microscopy showed robust penetration of CNS tissue by regenerating sensory axons treated with NT3 at 1 and 2 weeks postrhizotomy. Light and electron microscopical anterograde tracing experiments showed that these axons reentered appropriate and ectopic laminae of the dorsal horn, where they formed vesicle-filled synaptic buttons. Cord dorsum potential recordings confirmed that these were functional. In behavioral studies, NT3-treated (but not untreated or vehicle-treated) rats regained proprioception. Recovery depended on NT3-mediated sensory regeneration: preventing regeneration by root excision prevented recovery. NT3 treatment allows sensory axons to overcome inhibition present at the DREZ and may thus serve to promote functional recovery following dorsal root avulsions in humans.

Rho-Kinase Inhibition Enhances Axonal Plasticity and Attenuates Cold Hyperalgesia after Dorsal Rhizotomy

Dorsal rhizotomy results in primary deafferentation of the dorsal horn with concomitant sprouting of spared intraspinal monoaminergic axons. Because descending monoaminergic systems are thought to mitigate nociceptive transmission from the periphery and because dorsal rhizotomy can result in neuropathic pain, we sought to determine whether the rhizotomy-induced sprouting response could be further augmented. Because myelin-derived molecules mask endogenous plasticity of CNS axons and because myelin-inhibitory signaling occurs through the Rho-GTPase pathway, we inhibited Rho-pathway signaling after cervical dorsal rhizotomy in rats. An increase in the density of serotonergic- and tyrosine hydroxylase-positive fibers was seen in the dorsal horn 1 week after septuple rhizotomy, and axon density continued to increase for at least 1 month. One week after septuple rhizotomy, administration of intrathecal Y-27632, an antagonist of Rho-kinase (ROCK), increased the density of both fiber types over vehicle-treated controls. To examine behavioral effects of both cervical rhizotomy and ROCK inhibition, we examined responses to evoked pain: mechanical and thermal allodynia and cold hyperalgesia in the forepaw were examined after single, double, and quadruple rhizotomies of dorsal roots of the brachial plexus. The most notable behavioral outcome was the development of cold hyperalgesia in the affected forepaw after rhizotomies of the C7 and C8 dorsal roots. Application of Y-27632 both attenuated cold hyperalgesia and induced monoaminergic plasticity after C7/8 rhizotomy. Thus, inhibition of Rho-pathway signaling both promoted the sprouting of intact supraspinal monoaminergic fibers and alleviated pain after dorsal rhizotomy.

Nerve growth factor induces P2X(3) expression in sensory neurons.

Glial cell line‐derived neurotrophic factor (GDNF) and nerve growth factor (NGF) are neuroprotective for subpopulations of sensory neurons and thus are candidates for pain treatment. However, delivering these factors to damaged neurons will invariably result in undamaged systems also being treated, with possible consequences for sensory processing. In sensory neurons the purinergic receptor P2X3 is found predominantly in GDNF‐sensitive nociceptors. ATP signalling via the P2X3 receptor may contribute to pathological pain, suggesting an important role for this receptor in regulating nociceptive function. We therefore investigated the effects of intrathecal GDNF or NGF on P2X3 expression in adult rat spinal cord and dorsal root ganglia (DRG). In control spinal cords, P2X3 expression was restricted to a narrow band of primary afferent terminals within inner lamina II (IIi). Glial cell line‐derived neurotrophic factor treatment increased P2X3 immunoreactivity within lamina IIi but not elsewhere in the cord. Nerve growth factor treatment, however, induced novel P2X3 expression, with intense immunoreactivity in axons projecting to lamina I and outer lamina II and to the ventro‐medial afferent bundle beneath the central canal. In the normal DRG, we found a greater proportion of P2X3‐positive neurons at cervical levels, many of which were large‐diameter and calcitonin gene‐related peptide‐positive. In both cervical and lumbar DRG, the number of P2X3‐positive cells increased following GDNF or NGF treatment. De novo expression of P2X3 in NGF‐sensitive nociceptors may contribute to chronic inflammatory pain.

Peripherally-derived olfactory ensheathing cells do not promote primary afferent regeneration following dorsal root injury

Olfactory ensheathing cells (OECs) may support axonal regrowth, and thus might be a viable treatment for spinal cord injury (SCI); however, peripherally– derived OECs remain untested in most animal models of SCI. We have transplanted OECs from the lamina propria (LP) of mice expressing green fluorescent protein (GFP) in all cell types into immunosuppressed rats with cervical or lumbar dorsal root injuries. LP‐OECs were deposited into either the dorsal root ganglion (DRG), intact or injured dorsal roots, or the dorsal columns via the dorsal root entry zone (DREZ). LP‐OECs injected into the DRG or dorsal root migrated centripetally, and migration was more extensive in the injured root than in the intact root. These peripherally deposited OECs migrated within the PNS but did not cross the DREZ; similarly, large‐ or small‐caliber primary afferents were not seen to regenerate across the DREZ. LP‐OEC deposition into the dorsal columns via the DREZ resulted in a laminin‐rich injection track: due to the pipette trajectory, this track pierced the glia limitans at the DREZ. OECs migrated centrifugally through this track, but did not traverse the DREZ; axons entered the spinal cord via this track, but were not seen to reenter CNS tissue. We found a preferential association between CGRP‐positive small‐ to medium‐diameter afferents and OEC deposits in injured dorsal roots as well as within the spinal cord. In the cord, OEC deposition resulted in increased angiogenesis and altered astrocyte alignment. These data are the first to demonstrate interactions between sensory axons and peripherally– derived OECs following dorsal root injury.

Restoring function after spinal cord injury: towards clinical translation of experimental strategies

Spinal cord injury is currently incurable and treatment is limited to minimising secondary complications and maximising residual function by rehabilitation. Improved understanding of the pathophysiology of spinal cord injury and the factors that prevent nerve and tissue repair has fuelled a move towards more ambitious experimental treatments aimed at promoting neuroprotection, axonal regeneration, and neuroplasticity. By necessity, these new options are more invasive. However, in view of recent advances in spinal cord injury research and demand from patients, clinicians, and the scientific community to push promising experimental treatments to the clinic, momentum and optimism exist for the translation of candidate experimental treatments to clinical spinal cord injury. The ability to rescue, reactivate, and rewire spinal systems to restore function after spinal cord injury might soon be within reach.