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Despite the ability of peripheral nerves to regenerate after injury, failure occurs due to an inability of supporting cells to maintain growth, resulting in long-term consequences such as sensorimotor dysfunction and neuropathic pain. Here, we investigate the potential of engaging the cellular adaptive response to hypoxia, via inhibiting its negative regulators, to enhance the regenerative process. Under normoxic conditions, prolyl hydroxylase domain (PHD) proteins 1, 2, and 3 hydroxylate the key metabolic regulator hypoxia inducible factor 1α (HIF1α), marking it for subsequent proteasomal degradation. We inhibited PHD protein function systemically via either individual genetic deletion or pharmacological pan-PHD inhibition using dimethyloxalylglycine (DMOG). We show enhanced axonal regeneration after sciatic nerve crush injury in PHD1 -/- mice, PHD3 -/- mice, and in DMOG-treated mice, and in PHD1 -/- and DMOG-treated mice a reduction in hypersensitivity to cooling after permanent sciatic ligation. Electromyographically, PHD1 -/- and PHD3 -/- mice showed an increased CMAP amplitude one-month post-injury, probably due to protection against denervation induced muscle atrophy, while DMOG-treated and PHD2 +/- mice showed reduced latencies, indicating improved motor axon function. DMOG treatment did not affect the growth of dorsal root ganglion neurites in vitro, suggesting a lack of direct effects of DMOG on axonal regrowth. Enhanced regeneration in vivo was concurrent with an increase in macrophage density, and a shift in macrophage polarization state ratios (from M1-like toward M2-like) in DMOG-treated animals. These results indicate PHD proteins as a novel therapeutic target to improve regenerative and functional outcomes after peripheral nerve injury without manipulating molecular O 2 ., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Following spinal cord injury, spared axonal projections undergo spontaneous compensatory sprouting in an attempt to reinnervate synaptic targets that were deinnervated as a result of injury. However, compensatory sprouting is hindered by the expression of a myriad of inhibitory molecules throughout the adult central nervous system, including chondroitin sulfate proteoglycans (CSPGs) and myelin associated inhibitory proteins (MAIPs). In this study, we have identified a diketopiperazine designated DKP101516 that can overcome the inhibitory effects of MAIPs and CSPGs on neurite outgrowth and branching. In vivo analysis demonstrates that DKP101516 enhances the plasticity of various axonal populations following septuple dorsal rhizotomy by overcoming the inhibitory effects of CSPGs and MAIPs. Our results suggest that DKP101516 may encourage spinal cord repair by stimulating compensatory sprouting of intact axonal projections.

Bone morphogenetic proteins (BMPs) and their antagonists, including noggin, are required for nervous system development, but their potential roles in the reactions of the adult central nervous system to injury are unknown. Here we have examined the expression of noggin and BMPs in the spinal cord following dorsal rhizotomy. Through the use of a function-blocking antibody, we have also investigated the role of endogenous noggin in the neuritic plasticity which follows rhizotomy. Dorsal rhizotomy resulted in the upregulation of BMPs 2/4, 7 and noggin in the superficial white matter and in the dorsal neuropil of the spinal cord. These co-localized with glial fibrillary acidic protein, indicating their expression by astrocytes. Because BMPs induce dendritic sprouting and synaptogenesis in some neuronal populations in vitro, we hypothesized that administration of a noggin function-blocking antibody (FbAb) in vivo would augment rhizotomy-induced sprouting in the spinal cord. Topical application of noggin-FbAb to the dorsal surface of the spinal cord following rhizotomy resulted in significant increases in the density of microtubule-associated protein 2 (MAP-2) and substance P (SP)-positive processes within the lateral spinal nucleus. In the deafferented dorsal horn, noggin-FbAb treatment induced significant increases in the density of SP, calcitonin gene-related peptide (CGRP)- and 5-hydroxytryptamine (5-HT)-positive axons. These results suggest a novel mechanism by which endogenous plasticity of spared axons is suppressed following dorsal rhizotomy, and which might be exploited to improve the outcome of spinal cord injury and other CNS trauma.

After dorsal rhizotomy, sensory axons fail to regenerate beyond the astrocytic glia limitans at the dorsal root entry zone (DREZ) but this inhibition can be overcome with the delivery of exogenous neurotrophin-3. We investigated whether axonal inhibition at the DREZ is constitutive or induced after dorsal rhizotomy. Primary afferent neurones from enhanced green fluorescent protein-expressing mice were transplanted into adult rat dorsal root ganglia in the presence or absence of dorsal rhizotomy. In the absence of dorsal rhizotomy mouse axons freely extended into the rat central nervous system. After host dorsal rhizotomy, mouse axons were unable to cross the DREZ. However, in rats that received a dorsal rhizotomy concomitant with intrathecal neurotrophin-3, the mouse axons were able to cross the DREZ. These results indicate that, under normal circumstances, the adult DREZ is permissive to the regeneration of adult sensory axons and that it only becomes inhibitory once dorsal root axons have been injured and astrocytes at the DREZ have become reactive.

Myelin-derived molecules inhibit axonal regeneration in the CNS. The Long-Evans Shaker rat is a naturally occurring dysmyelinated mutant, which although able to express the components of myelin lacks functional myelin in adulthood. Given that myelin breakdown exposes axons to molecules that are inhibitory to regeneration, we sought to determine whether injured dorsal column axons in a Shaker rat would exhibit a regenerative response absent in normally myelinated Long-Evans (control) rats. Although Shaker rat axons did not regenerate beyond the lesion, they remained at the caudal end of the crush site. Control rat axons, in contrast, retracted and died back from the edge of the crush. The absence of retraction/dieback in Shaker rats was associated with a reduced phagocytic reaction to dorsal column crush around the caudal edge of the lesion. Systemic injection of minocycline, a tetracycline derivative, in control rats reduced both the macrophage response and axonal retraction/dieback following dorsal column injury. In contrast, increasing macrophage activation by spinal injection of the yeast particulate zymosan had no effect on axonal retraction/dieback in Shaker rats. Schwann cell invasion was reduced in minocycline-treated control rats compared with untreated control rats, and was almost undetectable in Shaker rats, suggesting that like axonal retraction/dieback, spinal Schwann cell infiltration is dependent upon macrophage-mediated myelin degeneration. These results indicate that following spinal cord injury the phagocyte-mediated degeneration of myelin and subsequent exposure of inhibitory molecules to the injured axons contributes to their retraction/dieback.

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.

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., ((C)2002 Elsevier Science (USA).)

Abstract: Neurons intrinsic to the spinal cord dorsal horn receive input from various classes of long-distance projection systems. Two of the best known of these are primary afferent and descending monoaminergic axons. Together with intrinsic interneurons, activity in these axonal populations shapes the early part of the sensory experience before it is transmitted to supraspinal structures via ascending projection axons. Injury to dorsal roots, which contain the centrally projecting branches of primary afferent axons, results in their permanent disconnection from the spinal cord, as well as sensory dysfunction such as pain. In animals, experimental dorsal root injuries affecting a small number of roots produce dynamic behavioural changes, providing evidence for the now familiar concept that sensory processing at the level of the spinal cord is not hard-wired. Changes in behaviour following rhizotomy suggest changes in spinal sensory circuitry, and we and others have shown that the density of spinal serotonergic axons as well as processes of inhibitory interneurons increases following rhizotomy. Intact primary afferent axons are less apt to sprout into denervated territory. Recent work from our group has asked (1) what is the stimulus that induces sprouting of serotonergic (and other) axons and (2) what prevents spared primary afferent axons from occupying the territory of those lost to injury. This article will review the evidence that a single factor upregulated by dorsal root injury, brain-derived neurotrophic factor (BDNF), underpins both serotonergic sprouting and a lack of primary afferent plasticity. BDNF also differentially modulates some of the behavioural consequences of dorsal root injury: antagonizing endogenous BDNF improves spontaneous mechanosensory recovery but prevents recovery from rhizotomy-induced hypersensitivity to cold. These findings reinforce the notion that in disease states as complex and variable as spinal cord injury, single pharmacological interventions are unlikely to produce meaningful results. However, understanding the differences in capacity for plasticity among different systems, as well as their triggers, should allow for more patient-tailored therapies. [Copyright &y& Elsevier]

Membranous compartments of neurons such as axons, dendrites and modified primary cilia are defining features of neuronal phenotype. This is unlike organelles deep to the plasma membrane, which are for the most part generic and not related directly to morphological, neurochemical or functional specializations. However, here we use multi-label immunohistochemistry combined with confocal and electron microscopy to identify a very large (∼6 microns in diameter), entirely intracellular neuronal organelle which occurs singly in a ubiquitous but neurochemically distinct and morphologically simple subset of sympathetic ganglion neurons. Although usually toroidal, it also occurs as twists or rods depending on its intracellular position: tori are most often perinuclear whereas rods are often found in axons. These 'loukoumasomes' (doughnut-like bodies) bind a monoclonal antibody raised against beta-III-tubulin (SDL.3D10), although their inability to bind other beta-III-tubulin monoclonal antibodies indicate that the responsible antigen is not known. Position-morphology relationships within neurons and their expression of non-muscle heavy chain myosin suggest a dynamic structure. They associate with nematosomes, enigmatic nucleolus-like organelles present in many neural and nonneural tissues, which we now show to be composed of filamentous actin. Loukoumasomes also separately interact with mother centrioles forming the basal body of primary cilia. They express gamma tubulin, a microtubule nucleator which localizes to non-neuronal centrosomes, and cenexin, a mother centriole-associated protein required for ciliogenesis. These data reveal a hitherto undescribed organelle, and depict it as an intracellular transport machine, shuttling material between the primary cilium, the nematosome, and the axon. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The dorsal root entry zone of the vagus nerve (vDREZ) is uniquely characterized by peripheral tissue insertions (PTIs) deep to the brainstem surface, consisting of Schwann cells and a reticulum of astrocytic processes. Because Schwann cells permit peripheral axonal regeneration, the capacity of vagal medullary PTIs to allow centripetal regeneration of visceral afferents after vagal dorsal rhizotomy in adult rats was investigated. The present work shows that vagal axons spontaneously regenerate into appropriate and ectopic brainstem nuclei. They accomplish this by first growing along PTIs but then extend along basal laminas of medullary blood vessels. Electrically stimulated regenerated vagal afferents induced Fos expression (indicating functional connectivity) within appropriate but not ectopic nuclei. The unique structure of the vDREZ can thus support spontaneous functional regeneration of visceral primary afferent axons into the adult CNS. [ABSTRACT FROM AUTHOR]

Abstract: The RIP monoclonal antibody is commonly used to identify oligodendrocytes. Recently, the RIP antigen was identified as 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), a known non-compact myelin protein [Watanabe, M., Sakurai, Y., Ichinose, T., Aikawa, Y., Kotani, M., Itoh, K., 2006. Monoclonal antibody Rip specifically recognizes 2′,3′-cyclic nucleotide 3′-phosphodiesterase in oligodendrocytes. J. Neurosci. Res. 84, 525–533]. In the present study we characterize normal and axotomy-induced changes in RIP immunoreactivity in peripheral glia. In myelinating Schwann cells, RIP demarcated paranodal regions of myelinated axons and clearly defined Schmidt–Lantermann incisures. Surprisingly, RIP immunoreactivity was not confined to myelinating glia. Robust RIP immunoreactivity was present in Remak bundles in mixed nerves and in sympathetic ganglia and grey rami. Following peripheral nerve injury, RIP immunoreactivity was redistributed diffusely throughout de-differentiating Schwann cell cytoplasm. In uninjured rats, low levels of RIP immunoreactivity were detectable in satellite cells surrounding dorsal root ganglion (DRG) neurons and in terminal Schwann cells at neuromuscular junctions. This pattern suggested a correlation between RIP immunoreactivity and the amount of axon–glial contact. We therefore injured the L5 spinal nerve to induce sympathetic sprouting and pericellular basket formation in the DRG, and asked whether relatively RIP-negative satellite glia, which normally contact only neuronal somata, would upregulate the RIP antigen upon contact with sprouting sympathetic axons. All perineuronal sympathetic sprouts infiltrated heavily RIP-immunoreactive satellite cell sheaths. RIP immunoreactivity was absent from placode-derived olfactory ensheathing glia, indicating that the relationship between axon–glial contact and RIP-immunoreactivity is restricted to peripheral ensheathing glia of the neural crest-derived Schwann cell lineage. [Copyright &y& Elsevier]

Abstract: Regeneration within or into the CNS is thwarted by glial inhibition at the site of a spinal cord injury and at the dorsal root entry zone (DREZ), respectively. At the DREZ, injured axons and their distal targets are separated by degenerating myelin and an astrocytic glia limitans. The different glial barriers to regeneration following dorsal rhizotomy are temporally and spatially distinct. The more peripheral astrocytic barrier develops first, and is surmountable by neurotrophin-3 (NT-3) treatment; the more central myelin-derived barrier, which prevents dorsal horn re-innervation by NT-3-treated axons, becomes significant only after the onset of myelin degeneration. Here we test the hypothesis that in the presence of NT-3, axonal regeneration is hindered by myelin degeneration products. To do so, we used the Long Evans Shaker (LES) rat, in which oligodendrocytes do not make CNS myelin, but do produce myelin-derived inhibitory proteins. We show that delaying NT-3 treatment for 1 week in normal (LE) rats, while allowing axonal penetration of the glia limitans and growth within degenerating myelin, results in misdirected regeneration with axons curling around presumptive degenerating myelin ovoids within the CNS compartment of the dorsal root. In contrast, delaying NT-3 treatment in LES rats resulted in straighter, centrally-directed regenerating axons. These results indicate that regeneration may be best optimized through a combination of neurotrophin treatment plus complete clearance of myelin debris. [Copyright &y& Elsevier]