MicroRNAs 21 and 199a-3p Regulate Axon Growth Potential through Modulation of Pten and mTor mRNAs.

Increased mTOR activity has been shown to enhance regeneration of injured axons by increasing neuronal protein synthesis, while PTEN signaling can block mTOR activity to attenuate protein synthesis. MicroRNAs (miRs) have been implicated in regulation of PTEN and mTOR expression, and previous work in spinal cord showed an increase in miR-199a-3p after spinal cord injury (SCI) and increase in miR-21 in SCI animals that had undergone exercise. Pten mRNA is a target for miR-21 and miR-199a-3p is predicted to target mTor mRNA. Here, we show that miR-21 and miR-199a-3p are expressed in adult dorsal root ganglion (DRG) neurons, and we used culture preparations to test functions of the rat miRs in adult DRG and embryonic cortical neurons. miR-21 increases and miR-199a-3p decreases in DRG neurons after in vivo axotomy. In both the adult DRG and embryonic cortical neurons, miR-21 promotes and miR-199a-3p attenuates neurite growth. miR-21 directly bound to Pten mRNA and miR-21 overexpression decreased Pten mRNA levels. Conversely, miR-199a-3p directly bound to mTor mRNA and miR-199a-3p overexpression decreased mTor mRNA levels. Overexpressing miR-21 increased both overall and intra-axonal protein synthesis in cultured DRGs, while miR-199a-3p overexpression decreased this protein synthesis. The axon growth phenotypes seen with miR-21 and miR-199a-3p overexpression were reversed by co-transfecting PTEN and mTOR cDNA expression constructs with the predicted 3' untranslated region (UTR) miR target sequences deleted. Taken together, these studies indicate that injury-induced alterations in miR-21 and miR-199a-3p expression can alter axon growth capacity by changing overall and intra-axonal protein synthesis through regulation of the PTEN/mTOR pathway.

Systemic Inhibition of Soluble Tumor Necrosis Factor with XPro1595 Exacerbates a Post-Spinal Cord Injury Depressive Phenotype in Female Rats.

Spinal cord injury (SCI) is associated with a three-fold risk of major depressive disorder compared with the general population. Current antidepressant therapy is often not as effective in this patient population, suggesting the need for a more efficacious therapeutic target. The goal of this study was to elucidate the role of inflammatory cytokine tumor necrosis factor (TNF) in the dorsal raphe nucleus (DRN, the principle source of serotonin to the brain) in the development and possible treatment of depression after SCI. A depressive phenotype following moderate T9 contusion was identified in adult female rats using a battery of behavioral tests (forced swim test, sucrose preference test, novel object recognition test, open field locomotion, and social exploration). Data revealed two clusters of injured rats (58%) that exhibit increased immobility in the forced swim test, indicating depressive phenotype or a melancholic-depressive phenotype with concomitant decrease in sucrose preference. ElevatedTNF levels in the DRN of these two clusters correlated with increased immobility in the forced swim test. We then tested the efficacy of soluble TNF inhibition with XPro1595 treatment to prevent the depressive phenotype after SCI. Subcutaneous (s.c.) delivery of XPro1595 caused an exacerbation of depressive phenotype, with all treated clusters exhibiting increased forced swim immobility compared with saline-treated non-depressed rats. Intracerebroventricular (i.c.v.) administration of the drug did not prevent or enhance the development of depression after injury. These results suggest a complex role for TNF-based neuroinflammation in SCI-induced depression that needs to be further explored, perhaps in conjunction with a broader targeting of additional post-SCI inflammatory cytokines.

Exercise-Induced Changes to the Macrophage Response in the Dorsal Root Ganglia Prevent Neuropathic Pain after Spinal Cord Injury.

Spinal cord injury (SCI) induces neuropathic pain that is refractory to treatment. Central and peripheral immune responses to SCI play critical roles in pain development. Although immune responses in the dorsal horn have been implicated in SCI-pain, immune mechanisms in the periphery, especially in the dorsal root ganglia (DRG), where nociceptor cell bodies reside, have not been well studied. Exercise is an immunomodulator, and we showed previously that early exercise after SCI reduces pain development. However, the mechanisms of exercise-mediated pain reduction are not understood. Therefore, we examined the 1) underlying immune differences in the spinal cord and DRG between rats with and without pain and 2) immunomodulatory effects of exercise in pain reduction. Rats were subjected to a unilateral contusion at C5 and tested for pain development using von Frey and mechanical conflict-avoidance paradigms. A subgroup of rats was exercised on forced running wheels starting at 5 days post-injury for 4 weeks. We observed greater microglial activation in the C7-C8 dorsal horn of rats with SCI-induced pain compared to rats with normal sensation, and early exercise reduced this activation independently of pain behavior. Further, abnormal pain sensation strongly correlated with an increased number of DRG macrophages. Importantly, exercise-treated rats that maintain normal sensation also have a lower number of macrophages in the DRG. Our data suggest that macrophage presence in the DRG may be an important effector of pain development, and early wheel walking exercise may mediate pain prevention by modulating the injury-induced macrophage response in the DRG. Further supportive evidence demonstrated that rats that developed pain despite exercise intervention still displayed a significantly elevated number of macrophages in the DRG. Collectively, these data suggest that macrophage presence in the DRG may be an amenable cellular target for future therapies.

Exercise and Peripheral Nerve Grafts as a Strategy To Promote Regeneration after Acute or Chronic Spinal Cord Injury.

Therapeutic interventions after spinal cord injury (SCI) routinely are designed to address multiple aspects of the primary and/or secondary damage that occurs. Exercise has a demonstrated efficacy for post-SCI complications such as cardiovascular dysfunction, neuropathic pain, and chronic inflammation, yet there is little understanding of the mechanisms by which improvements might result from this non-invasive approach. Here we review several of our observations of molecular and cellular changes within the injured spinal cord following acute or delayed exercise regimens that illustrate the potential for positive effects on neuroprotection and rehabilitation. Further, we provide new information about the role of exercise in promoting the regeneration of spinal axons into peripheral nerve grafts (PNGs) placed immediately or 6 weeks after injury. Acute and chronically injured propriospinal neurons within the lumbar spinal cord displayed the greatest propensity for enhanced regeneration after exercise, which correlates with the direct sensory input to this region from exercised hindlimb muscles. Future studies will extend these observations by testing whether exercise will boost the regenerative effort of axons to extend beyond the graft, interact with intraspinal targets, and establish functional connections across a lesion.

Intra-axonal protein synthesis - a new target for neural repair?

Although initially argued to be a feature of immature neurons with incomplete polarization, there is clear evidence that neurons in the peripheral nervous system retain the capacity for intra-axonal protein synthesis well into adulthood. This localized protein synthesis has been shown to contribute to injury signaling and axon regeneration in peripheral nerves. Recent works point to potential for protein synthesis in axons of the vertebrate central nervous system. mRNAs and protein synthesis machinery have now been documented in lamprey, mouse, and rat spinal cord axons. Intra-axonal protein synthesis appears to be activated in adult vertebrate spinal cord axons when they are regeneration-competent. Rat spinal cord axons regenerating into a peripheral nerve graft contain mRNAs and markers of activated translational machinery. Indeed, levels of some growth-associated mRNAs in these spinal cord axons are comparable to the regenerating sciatic nerve. Markers of active translation tend to decrease when these axons stop growing, but can be reactivated by a second axotomy. These emerging observations raise the possibility that mRNA transport into and translation within axons could be targeted to facilitate regeneration in both the peripheral and central nervous systems.

Dynamic Changes in Local Protein Synthetic Machinery in Regenerating Central Nervous System Axons after Spinal Cord Injury.

Intra-axonal localization of mRNAs and protein synthesis machinery (PSM) endows neurons with the capacity to generate proteins locally, allowing precise spatiotemporal regulation of the axonal response to extracellular stimuli. A number of studies suggest that this local translation is a promising target to enhance the regenerative capacity of damaged axons. Using a model of central nervous system (CNS) axons regenerating into intraspinal peripheral nerve grafts (PNGs) we established that adult regenerating CNS axons contain several different mRNAs and protein synthetic machinery (PSM) components in vivo. After lower thoracic level spinal cord transection, ascending sensory axons regenerate into intraspinal PNGs but axon growth is stalled when they reach the distal end of the PNG (3 versus 7 weeks after grafting, resp.). By immunofluorescence with optical sectioning of axons by confocal microscopy, the total and phosphorylated forms of PSMs are significantly lower in stalled compared with actively regenerating axons. Reinjury of these stalled axons increased axonal localization of the PSM proteins, indicative of possible priming for a subcellular response to axotomy. These results suggest that axons downregulate protein synthetic capacity as they cease growing, yet they retain the ability to upregulate PSM after a second injury.

Dopamine is produced in the rat spinal cord and regulates micturition reflex after spinal cord injury.

Dopamine (DA) neurons in the mammalian central nervous system are thought to be restricted to the brain. DA-mediated regulation of urinary activity is considered to occur through an interaction between midbrain DA neurons and the pontine micturition center. Here we show that DA is produced in the rat spinal cord and modulates the bladder reflex. We observed numerous tyrosine hydroxylase (TH)+ neurons in the autonomic nuclei and superficial dorsal horn in L6-S3 spinal segments. These neurons are dopamine-ß-hydroxylase (DBH)- and some contain detectable dopamine decarboxylase (DDC), suggesting their capacity to produce DA. Interestingly, following a complete thoracic spinal cord injury (SCI) to interrupt supraspinal projections, more TH+ neurons emerged in the lumbosacral spinal cord, coincident with a sustained, low level of DA expression there and a partially recovered micturition reflex. Non-selective blockade of spinal DA receptors reduced bladder activity whereas activation of spinal D2-like receptors increased bladder activity and facilitated voiding. Additionally, depletion of lumbosacral TH+ neurons with 6-hydroxydopamine (6-OHDA) decreased bladder non-voiding contractions and voiding efficiency. Furthermore, injecting the transsynaptic neuronal tracer pseudorabies virus (PRV) into the bladder detrusor labeled TH+ cells in the lumbosacral cord, confirming their involvement in spinal micturition reflex circuits. These results illustrate that DA is synthesized in the rat spinal cord; plasticity of lumbosacral TH+ neurons following SCI may contribute to DA expression and modulate the spinal bladder reflex. Thus, spinally-derived DA and receptors could be a novel therapeutic target to improve micturition recovery after SCI.

Delayed Exercise Is Ineffective at Reversing Aberrant Nociceptive Afferent Plasticity or Neuropathic Pain After Spinal Cord Injury in Rats.

Neuropathic pain is a debilitating consequence of spinal cord injury (SCI) that correlates with sensory fiber sprouting. Recent data indicate that exercise initiated early after SCI prevents the development of allodynia and modulated nociceptive afferent plasticity. This study determined if delaying exercise intervention until pain is detected would similarly ameliorate established SCI-induced pain. Adult, female Sprague-Dawley rats with a C5 unilateral contusion were separated into SCI allodynic and SCI non-allodynic cohorts at 14 or 28 days postinjury when half of each group began exercising on automated running wheels. Allodynia, assessed by von Frey testing, was not ameliorated by exercise. Furthermore, rats that began exercise with no allodynia developed paw hypersensitivity within 2 weeks. At the initiation of exercise, the SCI Allodynia group displayed marked overlap of peptidergic and non-peptidergic nociceptive afferents in the C7 and L5 dorsal horn, while the SCI No Allodynia group had scant overlap. At the end of 5 weeks of exercise both the SCI Allodynia and SCI No Allodynia groups had extensive overlap of the 2 c-fiber types. Our findings show that exercise therapy initiated at early stages of allodynia is ineffective at attenuating neuropathic pain, but rather that it induces allodynia-aberrant afferent plasticity in previously pain-free rats. These data, combined with our previous results, suggest that there is a critical therapeutic window when exercise therapy may be effective at treating SCI-induced allodynia and that there are postinjury periods when exercise can be deleterious.

Exercise dependent increase in axon regeneration into peripheral nerve grafts by propriospinal but not sensory neurons after spinal cord injury is associated with modulation of regeneration-associated genes.

Insufficient regeneration of central nervous system (CNS) axons contributes to persisting neurological dysfunction after spinal cord injury (SCI). Peripheral nerve grafts (PNGs) support regeneration by thousands of injured intraspinal axons and help them bypass some of the extracellular barriers that form after SCI. However this number represents but a small portion of the total number of axons that are injured. Here we tested if rhythmic sensory stimulation during cycling exercise would boost the intrinsic regenerative state of neurons to enhance axon regeneration into PNGs after a lower thoracic (T12) spinal transection of adult rats. Using True Blue retrograde tracing, we show that 4 weeks of cycling improves regeneration into a PNG from lumbar interneurons but not by primary sensory neurons. The majority of neurons that regenerate their axon are within 5 mm of the lesion and their number increased 70% with exercise. Importantly propriospinal neurons in more distant regions (5-20 mm from the lesion) that routinely exhibit very limited regeneration responded to exercise by increasing the number of regenerating neurons by 900%. There was no exercise-associated increase in regeneration from sensory neurons. Analyses using fluorescent in situ hybridization showed that this increase in regenerative response is associated with changes in levels of mRNAs encoding the regeneration associated genes (RAGs) GAP43, ß-actin and Neuritin. While propriospinal neurons showed increased mRNA levels in response to SCI alone and then to grafting and exercise, sensory neurons did not respond to SCI, but there was a response to the presence of a PNG. Thus, exercise is a non-invasive approach to modulate gene expression in injured neurons leading to an increase in regeneration. This sets the stage for future studies to test whether exercise will promote axon outgrowth beyond the PNG and reconnection with spinal cord neurons, thereby demonstrating a potential clinical application of this combined therapeutic intervention.

mRNAs and Protein Synthetic Machinery Localize into Regenerating Spinal Cord Axons When They Are Provided a Substrate That Supports Growth.

Although intra-axonal protein synthesis is well recognized in cultured neurons and during development in vivo, there have been few reports of mRNA localization and/or intra-axonal translation in mature CNS axons. Indeed, previous work indicated that mature CNS axons contain much lower quantities of translational machinery than PNS axons, leading to the conclusion that the capacity for intra-axonal protein synthesis is linked to the intrinsic capacity of a neuron for regeneration, with mature CNS neurons showing much less growth after injury than PNS neurons. However, when regeneration by CNS axons is facilitated, it is not known whether the intra-axonal content of translational machinery changes or whether mRNAs localize into these axons. Here, we have used a peripheral nerve segment grafted into the transected spinal cord of adult rats as a supportive environment for regeneration by ascending spinal axons. By quantitative fluorescent in situ hybridization combined with immunofluorescence to unambiguously distinguish intra-axonal mRNAs, we show that regenerating spinal cord axons contain ß-actin, GAP-43, Neuritin, Reg3a, Hamp, and Importin ß1 mRNAs. These axons also contain 5S rRNA, phosphorylated S6 ribosomal protein, eIF2α translation factor, and 4EBP1 translation factor inhibitory protein. Different levels of these mRNAs in CNS axons from regenerating PNS axons may relate to differences in the growth capacity of these neurons, although the presence of mRNA transport and likely local translation in both CNS and PNS neurons suggests an active role in the regenerative process. SIGNIFICANCE STATEMENT: Although peripheral nerve axons retain the capacity to locally synthesize proteins into adulthood, previous studies have argued that mature brain and spinal cord axons cannot synthesize proteins. Protein synthesis in peripheral nerve axons is increased during regeneration, and intra-axonally synthesized proteins have been shown to contribute to nerve regeneration. Here, we show that mRNAs and translational machinery are transported into axons regenerating from the spinal cord into the permissive environment of a peripheral nerve graft. Our data raise the possibility that spinal cord axons may make use of localized protein synthesis for regeneration.

Large animal and primate models of spinal cord injury for the testing of novel therapies.

Large animal and primate models of spinal cord injury (SCI) are being increasingly utilized for the testing of novel therapies. While these represent intermediary animal species between rodents and humans and offer the opportunity to pose unique research questions prior to clinical trials, the role that such large animal and primate models should play in the translational pipeline is unclear. In this initiative we engaged members of the SCI research community in a questionnaire and round-table focus group discussion around the use of such models. Forty-one SCI researchers from academia, industry, and granting agencies were asked to complete a questionnaire about their opinion regarding the use of large animal and primate models in the context of testing novel therapeutics. The questions centered around how large animal and primate models of SCI would be best utilized in the spectrum of preclinical testing, and how much testing in rodent models was warranted before employing these models. Further questions were posed at a focus group meeting attended by the respondents. The group generally felt that large animal and primate models of SCI serve a potentially useful role in the translational pipeline for novel therapies, and that the rational use of these models would depend on the type of therapy and specific research question being addressed. While testing within these models should not be mandatory, the detection of beneficial effects using these models lends additional support for translating a therapy to humans. These models provides an opportunity to evaluate and refine surgical procedures prior to use in humans, and safety and bio-distribution in a spinal cord more similar in size and anatomy to that of humans. Our results reveal that while many feel that these models are valuable in the testing of novel therapies, important questions remain unanswered about how they should be used and how data derived from them should be interpreted.

Exercise after spinal cord injury as an agent for neuroprotection, regeneration and rehabilitation.

Spinal cord injury (SCI) is a traumatic event from which there is limited recovery of function, despite the best efforts of many investigators to devise realistic therapeutic treatments. Partly this is due to the multifaceted nature of SCI, where there is considerable disarray and dysfunction secondary to the initial injury. Contributing to this secondary degeneration is neurotoxicity, vascular dysfunction, glial scarring, neuroinflammation, apoptosis and demyelination. It seems logical that addressing the need for neuroprotection, regeneration and rehabilitation will require different treatment strategies that may be applied at varied stages of the post-injury response. Here we focus on a single strategy, exercise/physical training, which appears to have multiple applications and benefits for an acute or chronic SCI. Exercise has been demonstrated to be advantageous at cellular and biochemical levels, as well as being of benefit for the whole animal or human subject. Data from our lab and others will be discussed to further elucidate the many positive aspects of implementing exercise following injury and to suggest that rehabilitation is not the sole target of a training regimen following SCI. This article is part of a Special Issue entitled SI: Spinal cord injury.

Either brain-derived neurotrophic factor or neurotrophin-3 only neurotrophin-producing grafts promote locomotor recovery in untrained spinalized cats.

Background. Transplants of cellular grafts expressing a combination of 2 neurotrophic factors, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been shown to promote and enhance locomotor recovery in untrained spinalized cats. Based on the time course of recovery and the absence of axonal growth through the transplants, we hypothesized that recovery was due to neurotrophin-mediated plasticity within the existing locomotor circuitry of the lumbar cord. Since BDNF and NT-3 have different effects on axonal sprouting and synaptic connectivity/strengthening, it becomes important to ascertain the contribution of each individual neurotrophins to recovery. Objective. We studied whether BDNF or NT-3 only producing cellular grafts would be equally effective at restoring locomotion in untrained spinal cats. Methods. Rat fibroblasts secreting one of the 2 neurotrophins were grafted into the T12 spinal transection site of adult cats. Four cats in each group (BDNF alone or NT-3 alone) were evaluated. Locomotor recovery was tested on a treadmill at 3 and 5 weeks post-transection/grafting. Results. Animals in both groups were capable of plantar weight-bearing stepping at speed up to 0.8 m/s as early as 3 weeks and locomotor capabilities were similar at 3 and 5 weeks for both types of graft. Conclusions. Even without locomotor training, either BDNF or NT-3 only producing grafts promote locomotor recovery in complete spinal animals. More clinically applicable delivery methods need to be developed.

Exercise modulates chloride homeostasis after spinal cord injury.

Activity-based therapies are routinely integrated in spinal cord injury (SCI) rehabilitation programs because they result in a reduction of hyperreflexia and spasticity. However, the mechanisms by which exercise regulates activity in spinal pathways to reduce spasticity and improve functional recovery are poorly understood. Persisting alterations in the action of GABA on postsynaptic targets is a signature of CNS injuries, including SCI. The action of GABA depends on the intracellular chloride concentration, which is determined largely by the expression of two cation-chloride cotransporters (CCCs), KCC2 and NKCC1, which serve as chloride exporters and importers, respectively. We hypothesized that the reduction in hyperreflexia with exercise after SCI relies on a return to chloride homeostasis. Sprague Dawley rats received a spinal cord transection at T12 and were assigned to SCI-7d, SCI-14d, SCI-14d+exercise, SCI-28d, SCI-28d+exercise, or SCI-56d groups. During a terminal experiment, H-reflexes were recorded from interosseus muscles after stimulation of the tibial nerve and the low-frequency-dependent depression (FDD) was assessed. We provide evidence that exercise returns spinal excitability and levels of KCC2 and NKCC1 toward normal levels in the lumbar spinal cord. Acutely altering chloride extrusion using the KCC2 blocker DIOA masked the effect of exercise on FDD, whereas blocking NKCC1 with bumetanide returned FDD toward intact levels after SCI. Our results indicate that exercise contributes to reflex recovery and restoration of endogenous inhibition through a return to chloride homeostasis after SCI. This lends support for CCCs as part of a pathway that could be manipulated to improve functional recovery when combined with rehabilitation programs.

Acute exercise prevents the development of neuropathic pain and the sprouting of non-peptidergic (GDNF- and artemin-responsive) c-fibers after spinal cord injury.

Spinal cord injury (SCI) impaired sensory fiber transmission leads to chronic, debilitating neuropathic pain. Sensory afferents are responsive to neurotrophic factors, molecules that are known to promote survival and maintenance of neurons, and regulate sensory neuron transduction of peripheral stimuli. A subset of primary afferent fibers responds only to the glial cell-line derived neurotrophic factor (GDNF) family of ligands (GFLs) and is non-peptidergic. In peripheral nerve injury models, restoration of GDNF or artemin (another GFL) to pre-injury levels within the spinal cord attenuates neuropathic pain. One non-invasive approach to increase the levels of GFLs in the spinal cord is through exercise (Ex), and to date exercise training is the only ameliorative, non-pharmacological treatment for SCI-induced neuropathic pain. The purpose of this study was 3-fold: 1) to determine whether exercise affects the onset of SCI-induced neuropathic pain; 2) to examine the temporal profile of GDNF and artemin in the dorsal root ganglia and spinal cord dorsal horn regions associated with forepaw dermatomes after SCI and Ex; and 3) to characterize GFL-responsive sensory fiber plasticity after SCI and Ex. Adult, female, Sprague-Dawley rats received a moderate, unilateral spinal cord contusion at C5. A subset of rats was exercised (SCI+Ex) on automated running wheels for 20min, 5days/week starting at 5days post-injury (dpi), continuing until 9 or 37dpi. Hargreaves' and von Frey testing was performed preoperatively and weekly post-SCI. Forty-two percent of rats in the unexercised group exhibited tactile allodynia of the forepaws while the other 58% retained normal sensation. The development of SCI-induced neuropathic pain correlated with a marked decrease in the levels of GDNF and artemin in the spinal cord and DRGs. Additionally, a dramatic increase in the density and the distribution throughout the dorsal horn of GFL-responsive afferents was observed in rats with SCI-induced allodynia. Importantly, in SCI rats that received Ex, the incidence of tactile allodynia decreased to 7% (1/17) and there was maintenance of GDNF and artemin at normal levels, with a normal distribution of GFL-responsive fibers. These data suggest that GFLs and/or their downstream effectors may be important modulators of pain fiber plasticity, representing effective targets for anti-allodynic therapeutics. Furthermore, we highlight the potent beneficial effects of acute exercise after SCI.

Axon regeneration and exercise-dependent plasticity after spinal cord injury.

Current dogma states that meaningful recovery of function after spinal cord injury (SCI) will likely require a combination of therapeutic interventions comprised of regenerative/neuroprotective transplants, addition of neurotrophic factors, elimination of inhibitory molecules, functional sensorimotor training, and/or stimulation of paralyzed muscles or spinal circuits. We routinely use (1) peripheral nerve grafts to support and direct axonal regeneration across an incomplete cervical or complete thoracic transection injury, (2) matrix modulation with chondroitinase (ChABC) to facilitate axonal extension beyond the distal graft-spinal cord interface, and (3) exercise, such as forced wheel walking, bicycling, or step training on a treadmill. We and others have demonstrated an increase in spinal cord levels of endogenous neurotrophic factors with exercise, which may be useful in facilitating elongation and/or synaptic activity of regenerating axons and plasticity of spinal neurons below the level of injury.

Exogenous BDNF enhances the integration of chronically injured axons that regenerate through a peripheral nerve grafted into a chondroitinase-treated spinal cord injury site.

Although axons lose some of their intrinsic capacity for growth after their developmental period, some axons retain the potential for regrowth after injury. When provided with a growth-promoting substrate such as a peripheral nerve graft (PNG), severed axons regenerate into and through the graft; however, they stop when they reach the glial scar at the distal graft-host interface that is rich with inhibitory chondroitin sulfate proteoglycans. We previously showed that treatment of a spinal cord injury site with chondroitinase (ChABC) allows axons within the graft to traverse the scar and reinnervate spinal cord, where they form functional synapses. While this improvement in outgrowth was significant, it still represented only a small percentage (<20%) of axons compared to the total number of axons that regenerated into the PNG. Here we tested whether providing exogenous brain-derived neurotrophic factor (BDNF) via lentivirus in tissue distal to the PNG would augment regeneration beyond a ChABC-treated glial interface. We found that ChABC treatment alone promoted axonal regeneration but combining ChABC with BDNF-lentivirus did not increase the number of axons that regenerated back into spinal cord. Combining BDNF with ChABC did increase the number of spinal cord neurons that were trans-synaptically activated during electrical stimulation of the graft, as indicated by c-Fos expression, suggesting that BDNF overexpression improved the functional significance of axons that did reinnervate distal spinal cord tissue.

Chronic at- and below-level pain after moderate unilateral cervical spinal cord contusion in rats.

Chronic neuropathic pain is a significant consequence of spinal cord injury (SCI) that is associated with evoked pain, including allodynia and/or hyperalgesia. Allodynia is defined as a painful response to normally innocuous stimuli, and hyperalgesia occurs when there is an amplified pain response to normally noxious stimuli. We describe a model of a unilateral cervical level (C5) contusion injury where sensory recovery was assessed weekly for 6 weeks in 32 adult, female, Sprague-Dawley rats. Bilateral thermal hyperalgesia and tactile allodynia are detectable in the fore- and hindpaws as early as 7 days post-injury (dpi) and persist for at least 42 days. Paw withdrawal latency in response to a noxious thermal stimulus significantly intra-animal pre-operative values. Change in paw withdrawal latency plateaued at 21 dpi. Interestingly, bilateral forepaw allodynia develops in fewer than 40% of rats as measured by von Frey monofilament testing. Similar results occur in the hindpaws, where bilateral allodynia occurs in 46% of rats with SCI. The contralesional forepaw and both hindpaws of rats showed a slight increase in paw withdrawal threshold to tactile stimuli acutely after SCI, corresponding to ipsilesional forelimb motor deficits that resolve over time. That there is no difference among allodynic and non-allodynic groups in overall spared tissue or specifically of the dorsal column or ventrolateral white matter where ascending sensory tracts reside suggests that SCI-induced pain does not depend solely on the size or extent of the lesion, but that other mechanisms are in play. These observations provide a valid model system for future testing of therapeutic interventions to prevent the onset or to reduce the debilitating effects of chronic neuropathic pain after SCI.

Plasticity in ascending long propriospinal and descending supraspinal pathways in chronic cervical spinal cord injured rats.

The high clinical relevance of models of incomplete cervical spinal cord injury (SCI) creates a need to address the spontaneous neuroplasticity that underlies changes in functional activity that occur over time after SCI. There is accumulating evidence supporting long projecting propriospinal neurons as suitable targets for therapeutic intervention after SCI, but focus has remained primarily oriented toward study of descending pathways. Long ascending axons from propriospinal neurons at lower thoracic and lumbar levels that form inter-enlargement pathways are involved in forelimb-hindlimb coordination during locomotion and are capable of modulating cervical motor output. We used non-invasive magnetic stimulation to assess how a unilateral cervical (C5) spinal contusion might affect transmission in intact, long ascending propriospinal pathways, and influence spinal cord plasticity. Our results show that transmission is facilitated in this pathway on the ipsilesional side as early as 1 week post-SCI. We also probed for descending magnetic motor evoked potentials (MMEPs) and found them absent or greatly reduced on the ipsilesional side as expected. The frequency-dependent depression (FDD) of the H-reflex recorded from the forelimb triceps brachii was bilaterally decreased although H(max)/M(max) was increased only on the ipsilesional side. Behaviorally, stepping recovered, but there were deficits in forelimb-hindlimb coordination as detected by BBB and CatWalk measures. Importantly, epicenter sparing correlated to the amplitude of the MMEPs and locomotor recovery but it was not significantly associated with the inter-enlargement or segmental H-reflex. In summary, our results indicate that complex plasticity occurs after a C5 hemicontusion injury, leading to differential changes in ascending vs. descending pathways, ipsi- vs. contralesional sides even though the lesion was unilateral as well as cervical vs. lumbar local spinal networks.

Nanofibrous collagen nerve conduits for spinal cord repair.

Nerve regeneration in an injured spinal cord is often restricted, contributing to the devastating outcome of neurologic impairment below the site of injury. Although implantation of tissue-engineered scaffolds has evolved as a potential treatment method, the outcomes remain sub-optimal. One possible reason may be the lack of topographical signals from these constructs to provide contact guidance to invading cells or regrowing axons. Nanofibers mimic the natural extracellular matrix architecturally and may therefore promote physiologically relevant cellular phenotypes. In this study, the potential application of electrospun collagen nanofibers (diameter=208.2±90.4 nm) for spinal cord injury (SCI) treatment was evaluated in vitro and in vivo. Primary rat astrocytes and dorsal root ganglias (DRGs) were seeded on collagen-coated glass cover slips (two-dimensional [2D] substrate controls), and randomly oriented or aligned collagen fibers to evaluate scaffold topographical effects on astrocyte behavior and neurite outgrowth, respectively. When cultured on collagen nanofibers, astrocyte proliferation and expression of glial fibrillary acidic protein (GFAP) were suppressed as compared to cells on 2D controls at days 3 (p<0.05) and 7 (p<0.01). Aligned fibers resulted in elongated astrocytes (elongation factor >4, p<0.01) and directed the orientation of neurite outgrowth from DRGs along fiber axes. In the contrast, neurites emanated radially on randomly oriented collagen fibers. By forming collagen scaffolds into spiral tubular structures, we demonstrated the feasibility of using electrospun nanofibers for the treatment of acute SCI using a rat hemi-section model. At days 10 and 30 postimplantation, extensive cellular penetration into the constructs was observed regardless of fiber orientation. However, scaffolds with aligned fibers appeared more structurally intact at day 30. ED1 immunofluorescent staining revealed macrophage invasion by day 10, which decreased significantly by day 30. Neural fiber sprouting as evaluated by neurofilament staining was observed as early as day 10. In addition, GFAP immunostained astrocytes were found only at the boundary of the lesion site, and no astrocyte accumulation was observed in the implantation area at any time point. These findings indicate the feasibility of fabricating 3D spiral constructs using electrospun collagen fibers and demonstrated the potential of these scaffolds for SCI repair.