Search

Abstract CSPGs are components of the extracellular matrix in the nervous system, where they serve as cues for axon guidance during development. After a peripheral nerve injury, CSPGs switch roles and become axon inhibitors and become diffusely distributed at the injury site. To investigate whether the spatial distribution of CSPGs affects their role, we combined in vitro DRG cultures with CSPG stripe or coverage assays to simulate the effect of a patterned substrate or dispersive distribution of CSPGs on growing neurites. We observed neurite steering at linear CSPG interfaces and neurite inhibition when diffused CSPGs covered the distal but not the proximal segment of the neurite. The repellent and inhibitory effects of CSPGs on neurite outgrowth were associated with the disappearance of focal actin filaments on growth cones. The application of an actin polymerization inducer, jasplakinolide, allowed neurites to break through the CSPG boundary and grow on CSPG-coated surfaces. The results of our study collectively reveal a novel mechanism that explains how the spatial distribution of CSPGs determines whether they act as a cue for axon guidance or as an axon-inhibiting factor. Increasing our understanding of this issue may promote the development of novel therapeutic strategies that regulate the spatial distributions of CSPGs to use them as an axon guidance cue. Highlights • Different spatial distributions of CSPGs induce different neurite regeneration behaviors. • CSPGs have no inhibitory effect on distal neurite when acting on proximal neurite. • Actin filaments redistribute repulsively when CSPGs interact with growth cone. • Stabilization of actin filaments in growth cone reduces the axon inhibitory effect of CSPGs. [ABSTRACT FROM AUTHOR]

Activity dependent plasticity is a key mechanism for the central nervous system (CNS) to adapt to its environment. Whether neuronal activity also influences axonal regeneration in the injured CNS, and whether electrical stimulation (ES) can activate regenerative programs in the injured CNS remains incompletely understood. Using KCl-induced depolarization, in vivo ES followed by ex-vivo neurite growth assays and ES after spinal cord lesions and cell grafting, we aimed to identify parameters important for ES-enhanced neurite growth and axonal regeneration. Using cultures of sensory neurons, neurite growth was analyzed after KCl-induced depolarization for 1-72 h. Increased neurite growth was detected after short-term stimulation and after longer stimulation if a sufficient delay between stimulation and growth measurements was provided. After in vivo ES (20 Hz, 2 × motor threshold, 0.2 ms, 1 h) of the intact sciatic nerve in adult Fischer344 rats, sensory neurons showed a 2-fold increase in in vitro neurite length one week later compared to sham animals, an effect not observed one day after ES. Longer ES (7 h) and repeated ES (7 days, 1 h each) also increased growth by 56–67% one week later, but provided no additional benefit. In vivo growth of dorsal column sensory axons into a graft of bone marrow stromal cells 4 weeks after a cervical spinal cord lesion was also enhanced with a single post-injury 1 h ES of the intact sciatic nerve and was also observed after repeated ES without inducing pain-like behavior. While ES did not result in sensory functional recovery, our data indicate that ES has time-dependent influences on the regenerative capacity of sensory neurons and might further enhance axonal regeneration in combinatorial approaches after SCI. [ABSTRACT FROM AUTHOR]

Although previous studies have identified several strategies to stimulate regeneration of CNS axons, extensive regeneration and functional recovery have remained a major challenge, particularly for large diameter myelinated axons. Within the CNS, myelin is thought to inhibit axon regeneration, while modulating activity of the mTOR pathway promotes regeneration of injured axons. In this study, we examined NT-3 mediated regeneration of sensory axons through the dorsal root entry zone in a triple knockout of myelin inhibitory proteins or after activation of mTOR using a constitutively active (ca) Rheb in DRG neurons to determine the influence of environmental inhibitory or activation of intrinsic growth pathways could enhance NT-3-mediate regeneration. Loss of myelin inhibitory proteins showed modest enhancement of sensory axon regeneration. In mTOR studies, we found a dramatic age related decrease in the mTOR activation as determined by phosphorylation of the downstream marker S6 ribosomal subunit. Expression of caRheb within adult DRG neurons in vitro increased S6 phosphorylation and doubled the overall length of neurite outgrowth, which was reversed in the presence of rapamycin. In adult female rats, combined expression of caRheb in DRG neurons and NT-3 within the spinal cord increased regeneration of sensory axons almost 3 fold when compared to NT-3 alone. Proprioceptive assessment using a grid runway indicates functionally significant regeneration of large-diameter myelinated sensory afferents. Our results indicate that caRheb-induced increase in mTOR activation enhances neurotrophin-3 induced regeneration of large-diameter myelinated axons. [ABSTRACT FROM AUTHOR]

After spinal cord injury (SCI) in mammals, injured axons fail to regenerate. By contrast, lampreys recover from complete spinal transection and axons regenerate selectively in their correct paths. Yet the large, identified reticulospinal neurons in the lamprey brain vary greatly in their regenerative abilities – some have high regeneration capacity (probability of regeneration > 50%) and others have low regeneration capacity (< 30%) – even though they have similar projection paths. The presence of both regenerating and non-regenerating neurons located in the same brain region and projecting to the same axon tracts suggests that differences in their regenerating abilities depend upon factors intrinsic to the neurons. Previous work has suggested that axon regeneration, especially in PNS, could depend on epigenetic mechanisms of histone modifications, such as the acetylation of histone tails. Our data indicated that expression of the enzymes responsible for regulating the acetylation of histone (KATs and HDACs) - KAT2A, KAT5 and P300 and HDAC3 did not change after SCI in either high regeneration capacity or low regeneration capacity neurons. In the present report, we show a novel and unexpected relationship between neuron regeneration abilities and expression of HDAC1. While HDAC1 expression was downregulated in both high and low regeneration capacity neurons 2 and 4 weeks after SCI, it was upregulated at 7 weeks at almost all RS neurons. However, at 10 weeks post-transection only high regeneration capacity neurons displayed elevated HDAC1 mRNA expression and HDAC1 expression was again downregulated in low regeneration capacity neurons. Moreover, we show that HDAC1 is preferentially expressed in regenerated neurons, but not in non-regenerating neurons. Together, these results suggest that SCI causes significant changes in HDAC1 expression and that HDAC1 expression in regenerating neurons may modulates a survival or regeneration programs. [ABSTRACT FROM AUTHOR]

Peripheral nerve (PN) grafts can be used to bridge tissue defects in the CNS. Using a PN-to-optic nerve (ON) graft model, we combined gene therapy with pharmacotherapy to promote the long-distance regeneration of injured adult retinal ganglion cells (RGCs). Autologous sciatic nerve was sutured onto the transected ON and the distal end immediately inserted into contralateral superior colliculus (SC). Control rats received intraocular injections of saline or adeno-associated virus (AAV) encoding GFP. In experimental groups, three bi- cis tronic AAV vectors encoding ciliary neurotrophic factor (CNTF) were injected into different regions of the grafted eye. Each vector encoded a different fluorescent reporter to assess retinotopic order in the regenerate projection. To encourage sprouting/synaptogenesis, after 6 weeks some AAV–CNTF injected rats received an intravitreal injection of recombinant brain-derived neurotrophic factor (rBDNF) or AAV–BDNF. Four months after surgery, cholera toxin B was used to visualize regenerate RGC axons. RGC viability and axonal regrowth into SC were significantly greater in AAV–CNTF groups. In some cases, near the insertion site, regenerate axonal density resembled retinal terminal densities seen in normal SC. Complex arbors were seen in superficial but not deep SC layers and many terminals were immunopositive for presynaptic proteins vGlut2 and SV2. There was improvement in visual function via the grafted eye with significantly greater pupillary constriction in both AAV–CNTF + BDNF groups. In both control and AAV–CNTF + rBDNF groups the extent of light avoidance correlated with the maximal distance of axonal penetration into superficial SC. Despite the robust regrowth of RGC axons back into the SC, axons originating from different parts of the retina were intermixed at the PN graft/host SC interface, indicating that there remained a lack of order in this extensive regenerate projection. [ABSTRACT FROM AUTHOR]

Chondroitin Sulfate Proteoglycans (CSPGs) are a major component of the extracellular matrix in the central nervous system (CNS) and play critical role in the development and pathophysiology of the brain and spinal cord. Developmentally, CSPGs provide guidance cues for growth cones and contribute to the formation of neuronal boundaries in the developing CNS. Their presence in perineuronal nets plays a crucial role in the maturation of synapses and closure of critical periods by limiting synaptic plasticity. Following injury to the CNS, CSPGs are dramatically upregulated by reactive glia which form a glial scar around the lesion site. Increased level of CSPGs is a hallmark of all CNS injuries and has been shown to limit axonal plasticity, regeneration, remyelination, and conduction after injury. Additionally, CSPGs create a non-permissive milieu for cell replacement activities by limiting cell migration, survival and differentiation. Mounting evidence is currently shedding light on the potential benefits of manipulating CSPGs in combination with other therapeutic strategies to promote spinal cord repair and regeneration. Moreover, the recent discovery of multiple receptors for CSPGs provides new therapeutic targets for targeted interventions in blocking the inhibitory properties of CSPGs following injury. Here, we will provide an in depth discussion on the impact of CSPGs in normal and pathological CNS. We will also review the recent preclinical therapies that have been developed to target CSPGs in the injured CNS. [ABSTRACT FROM AUTHOR]

While it is well established that the axons of adult neurons have a lower capacity for regrowth, some regeneration of certain CNS populations after spinal cord injury (SCI) is possible if their axons are provided with a permissive substrate, such as an injured peripheral nerve. While some axons readily regenerate into a peripheral nerve graft (PNG), these axons almost always stall at the distal interface and fail to reinnervate spinal cord tissue. Treatment of the glial scar at the distal graft interface with chondroitinase ABC (ChABC) can improve regeneration, but most regenerated axons need further stimulation to extend beyond the interface. Previous studies demonstrate that pharmacologically inhibiting kinesin-5, a motor protein best known for its essential role in mitosis but also expressed in neurons, with the pharmacological agent monastrol increases axon growth on inhibitory substrates in vitro . We sought to determine if monastrol treatment after an SCI improves functional axon regeneration. Animals received complete thoracic level 7 (T7) transections and PNGs and were treated intrathecally with ChABC and either monastrol or DMSO vehicle. We found that combining ChABC with monastrol significantly enhanced axon regeneration. However, there were no further improvements in function or enhanced c-Fos induction upon stimulation of spinal cord rostral to the transection. This indicates that monastrol improves ChABC-mediated axon regeneration but that further treatments are needed to enhance the integration of these regrown axons. [ABSTRACT FROM AUTHOR]

Abstract: Neurotrophins and the transplantation of bone marrow-derived stromal cells (MSCs) are both candidate therapies targeting spinal cord injury (SCI). While some studies have suggested the ability of MSCs to transdifferentiate into neural cells, other SCI studies have proposed anti-inflammatory and other mechanisms underlying established beneficial effects. We grafted rat MSCs genetically modified to express MNTS1, a multineurotrophin that binds TrkA, TrkB and TrkC, and p75NTR receptors or MSC-MNTS1/p75− that binds mainly to the Trk receptors. Seven days after contusive SCI, PBS-only, GFP-MSC, MSC-MNTS1/GFP or MSC-MNTS1/p75−/GFP were delivered into the injury epicenter. All transplanted groups showed reduced inflammation and cystic cavity size compared to control SCI rats. Interestingly, transplantation of the MSC-MNTS1 and MSC-MNTS1/p75−, but not the naïve MSCs, enhanced axonal growth and significantly prevented cutaneous hypersensitivity after SCI. Moreover, transplantation of MSC-MNTS1/p75− promoted angiogenesis and modified glial scar formation. These findings suggest that MSCs transduced with a multineurotrophin are effective in promoting cell growth and improving sensory function after SCI. These novel data also provide insight into the neurotrophin-receptor dependent mechanisms through which cellular transplantation leads to functional improvement after experimental SCI. [Copyright &y& Elsevier]

Abstract: Adult neurons of the peripheral nervous system (PNS), in contrast to those of the central nervous system, have a remarkable capacity to repair themselves after injury, yet the mechanisms underlying this regenerative propensity of peripheral neurons are far from completely understood. Here we show that the transcription factor CCAAT enhancer binding protein delta (C/EBPδ) is necessary for the efficient axonal regeneration of dorsal root ganglia (DRG) neurons after sciatic nerve crush injury. Loss of C/EBPδ substantially impairs axonal growth in dissociated cultured DRG neurons. In addition, lack of C/EPBδ causes a major reduction in the regenerative response of DRG neurons to a conditioning lesion, which is a well known paradigm of injury that enhances axonal growth due to a transcription-dependent cell body response. C/EBPδ is required for the induction of selected regeneration-associated genes. For example, the expression of SPRR1A (small proline-rich repeat protein 1A) is greatly reduced in DRG neurons of C/EBPδ knockout mice during axonal regeneration compared to those in wild-type mice, while the expression of GAP-43 (growth associated protein-43) and galanin is not affected. Nevertheless, the expected prompt recovery of sciatic nerve function after injury is severely impaired in C/EBPδ knockout mice, having a delay time of approximately 1month for reaching the full function of recovering wild-type mice, suggesting that a transcription mechanism mediated by C/EBPδ is required for efficient axonal regeneration. Taken together, our results identify C/EBPδ as a crucial component of the transcriptional regulatory machinery which underlies the intrinsic capacity of peripheral neurons for axonal regeneration. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: De-focused low energy extracorporeal shock wave therapy (ESWT) has been widely used in various clinical and experimental models for the treatment of painful conditions such as epicondylitis and plantar fascitis and also bone and wound healing. There is evidence that ESWT improves the metabolic activity of various cell types, e.g. chondrocytes and endothelial cells but little is known about its effects on nervous tissue. The aim of this study was to investigate whether ESWT improves the regeneration of injured nerves in an experimental rat model. Sprague–Dawley rats received an 8mm long homotopic nerve autograft into the right sciatic nerve, fixed with epineurial sutures. Two experimental groups were set up: the group 1 animals received ESWT (300 impulses, 3Hz) immediately after nerve grafting whereas the group 2 (control) animals received only nerve autografts. Serial CatWalk automated gait analysis, electrophysiological studies and morphological investigations were carried out. The survival time was either 3weeks or 3months. At 6 to 8weeks of survival the ESWT group of animals exhibited a significantly improved functional recovery relative to the controls. Electrophysiological observations at 3weeks after surgery revealed marked values of amplitude (3.9±0.8mV, S.E.M.) and compound nerve action potential (CNAP, 5.9±1.4mV·ms, S.E.M.) in the ESWT group, whereas there were no detectable amplitudes in the control group. This finding was accompanied by significantly greater numbers of myelinated nerve fibres in the middle of the graft (4644±170 [S.E.M., ESWT] vs 877±68 [S.E.M., control]) and in the distal stump (1586±157 [S.E.M., ESWT] vs 308±29 [S.E.M., control]) of ESWT animals relative to the controls 3weeks after surgery. Three weeks after surgery the nerve grafts of control animals contained great numbers of phagocytes and unmyelinated nerve fibres, while the ESWT nerve grafts were filled with well-myelinated regenerating axons. There was no significant difference between the numbers of endoneural vessels in the ESWT and the control nerves. Three months after surgery, no significant differences were observed in the functional and electrophysiological data. Equally high numbers of myelinated axons distal to the graft could be found in both groups (7693±673 [S.E.M., ESWT] vs 6090±716 [S.E.M., control]). These results suggest that ESWT induces an improved rate of axonal regeneration, this phenomenon probably involving faster Wallerian degeneration, the improved removal of degenerated axons and a greater capacity of the injured axons to regenerate. [Copyright &y& Elsevier]

Abstract: This study investigated whether neuronal inhibitor of DNA binding 2 (Id2), a regulator of basic helix-loop-helix (bHLH) transcription factors, can activate the intrinsic neuritogenetic mode of dorsal root ganglion (DRG) neurons in adult mice following spinal cord injury (SCI). First, the Id2 developmental expression profile of DRG neurons, along with the correlated activity of Cdh1-anaphase promoting complex (Cdh1-APC), was characterized. Next, a D-box mutant Id2 (Id2DBM) adenoviral vector, resistant to Cdh1-APC degradation, was developed to enhance neuronal Id2 expression. After the vector was introduced into DRG neurons, the effect of Id2 on neurite outgrowth of cultured DRG neurons and sensory axonal regeneration following spinal cord dorsal hemisection was evaluated. The expression of Id2 in DRG neurons was high in the embryonic stage, downregulated after birth, and significantly reduced in the adult. Expression of Cdh1-APC was opposite to Id2, which may be responsible for Id2 degradation during DRG maturation. Overexpression of Id2DBM in DRG neurons enhanced neuritogenesis on both permissive and inhibitory substrates. Following spinal cord dorsal hemisection, overexpression of Id2DBM reduced axon dieback and increased the number and length of regenerative fibers into the lesion gap. Reprogramming the intrinsic growth status of quiescent adult DRG neurons by enhancing Id2 expression results in active neuritogenesis following SCI. Id2 may be a novel target for enhancing sensory axonal regeneration following injuries to the adult spinal cord. [Copyright &y& Elsevier]

Abstract: Reactive astrogliosis impedes axonal regeneration after injuries to the mammalian central nervous system (CNS). Here we report that glial cell line-derived neurotrophic factor (GDNF), combined with transplanted Schwann cells (SCs), effectively reversed the inhibitory properties of astrocytes at graft–host interfaces allowing robust axonal regeneration, concomitant with vigorous migration of host astrocytes into SC-seeded semi-permeable guidance channels implanted into a right-sided spinal cord hemisection at the 10th thoracic (T10) level. Within the graft, migrated host astrocytes were in close association with regenerated axons. Astrocyte processes extended parallel to the axons, implying that the migrated astrocytes were not inhibitory and might have promoted directional growth of regenerated axons. In vitro, GDNF induced migration of SCs and astrocytes toward each other in an astrocyte–SC confrontation assay. GDNF also enhanced migration of astrocytes on a SC monolayer in an inverted coverslip migration assay, suggesting that this effect is mediated by direct cell–cell contact between the two cell types. Morphologically, GDNF administration reduced astrocyte hypertrophy and induced elongated process extension of these cells, similar to what was observed in vivo. Notably, GDNF treatment significantly reduced production of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs), two hallmarks of astrogliosis, in both the in vivo and in vitro models. Thus, our study demonstrates a novel role of GDNF in modifying spinal cord injury (SCI)-induced astrogliosis resulting in robust axonal regeneration in adult rats. [Copyright &y& Elsevier]

Abstract: The extensive period of retinal ganglion cell (RGC) neurogenesis in the rat is associated with a protracted sequence of arrival of their axons into central targets such as the superior colliculus (SC) (Dallimore et al., 2002). Using in utero 5-bromo-2''-deoxyrudine (BrdU) injections to label early (embryonic day (E) 15) or late (E18 or E19) born RGCs, we now show that E15 RGCs with axons that enter the SC prenatally undergo programmed cell death earlier than late-born RGCs whose axons only reach the SC late in the first postnatal week. These late-born RGCs do not begin to die until postnatal day (P) 5/6. Removal of retrograde trophic support by P1 SC ablation initially only affects E15 RGCs; however by P5 death of late-born RGCs is increased, confirming that a switch to target dependency is delayed in this cohort. In a further experiment it was found that, following complete rostral SC transection at P2, the proportion of post-lesion axons originating from E19 RGCs was significantly greater than the proportion that normally makes up the retinotectal projection. Thus, even in neonatal brain, uninjured late-arriving axons are more likely to grow across a lesion site than injured axons undergoing regeneration. To study if birth date also affects regenerative potential in adulthood, autologous peripheral nerve (PN) was grafted onto the cut optic nerve in mature BrdU labelled rats. We found that, compared to E15 RGCs, a significantly greater proportion of late-born RGCs survived axotomy, but comparatively fewer of these surviving E19 RGCs regrew an axon into a graft. In summary, this research shows that the birthdate of RGCs significantly impacts on their subsequent life history and response to injury. Understanding how developing central nervous system (CNS) neurons acquire dependency on target-derived trophic support may lead to new strategies for enhancing survival and regeneration in adult CNS. [Copyright &y& Elsevier]

Abstract: Reimplantation of avulsed rat lumbar spinal ventral roots results in poor recovery of function of the denervated hind limb muscles. In contrast, reimplantation of cervical or sacral ventral roots is a successful repair strategy that results in a significant degree of regeneration. A possible explanation for this difference could be that following lumbar root avulsion, axons have to travel longer distances towards their target muscles, resulting in prolonged denervation of the distal nerve and a diminished capacity to support regeneration. Here we present a detailed spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression following unilateral avulsion and implantation of lumbar ventral roots L3, L4, and L5. Reimplantation prolongs the survival of motoneurons up to one month post-lesion. The first regenerating motor axons entered the reimplanted ventral roots during the first week and large numbers of fibers gradually enter the lumbar plexus between 2 and 4 weeks, indicating that axons enter the reimplanted roots and plexus over an extended period of time. However, motor axon counts show that relatively few axons reach the distal sciatic nerve in the 16 week post-lesion period. The observed initial increase and subsequent decline in expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor correlate with the apparent spatio-temporal decline in the regenerative capacity of motor axons, indicating that the distal nerve is losing its capacity to support regenerating motor axons following prolonged denervation. These findings have important implications for future strategies to promote long-distance regeneration through distal, chronically denervated peripheral nerves. [Copyright &y& Elsevier]

Abstract: Peripheral nerve injuries induce plastic changes on primary afferent fibers and on the spinal circuitry, which are related to the emergence of neuropathic pain. In this study we compared three models of sciatic nerve injury in the rat with different degrees of damage and impact on regeneration capability: crush nerve injury, chronic constriction injury (CCI) and spared nerve injury (SNI). All three models were characterized by means of nerve histology, in order to describe the degenerative and regenerative process of injured axons. Nociceptive responses were evaluated by mechanical and thermal algesimetry tests. Crush animals displayed higher withdrawal thresholds on the ipsilateral paw compared to the contralateral during the time of denervation, while CCI and SNI animals showed mechanical and thermal hyperalgesia. Central plasticity was evaluated by immunohistochemical labeling of non-peptidergic (IB4-positive) and peptidergic (substance P-positive) nociceptive C-fibers on L4–L6 spinal cord sections. After crush nerve injury and SNI, we observed progressive and sustained reduction of IB4 and SP immunolabeling at the sciatic projection territory in the superficial laminae of the dorsal horn, which affected only the tibial and peroneal nerves projection areas in the case of SNI. After CCI, changes on SP-immunoreactivity were not observed, and IB4-immunoreactive area decreased initially but recovered to normal levels on the second week post-injury. Thus, nociceptive responses depend on the type of injury, and the immunoreactivity pattern of afferent fibers at the spinal cord display changes less pronounced after partial than complete sciatic nerve injury. Although signs of neuropathic pain appear in all three lesion models, nociceptive responses and central plasticity patterns differ between them. [Copyright &y& Elsevier]

Abstract: Reconnection of interrupted peripheral nerve by microsurgical suture is a common clinical practice. However, the extent to which peripheral neurorrhaphy improves nerve regeneration and functional recovery remains unsatisfactory. Here, we used anatomical and electrophysiological techniques to investigate the temporal correlation between the expressions of oxidative stress-related biomarkers such as neuronal nitric oxide synthase (nNOS) and the facial axonal regeneration after an immediate facial nerve repair in adult rats since peripheral nerve lesion is well known to induce a dramatic increase of NOS expression in the affected neuronal cell bodies. We found that compared to nerve cut without suture, facial nerve repair not only caused the facial axonal regeneration but also consistently prevented the fluctuations of expressions of oxidative stress-related biomarkers in 10 weeks postlesion. To further elucidate the role of nitric oxide (NO) in the axonal degeneration/regeneration, four different NOS inhibitors were applied to additional rats after facial nerve cut or repair. Both of facial nerve cut+NOS inhibition and facial nerve repair+NOS inhibition were seen to prevent the alterations of expressions of the biomarkers, no matter which NOS inhibitor was used. Moreover, we found that facial nerve repair+NOS inhibition promoted earlier and better axonal regeneration than facial nerve repair, demonstrated by labeling of neuromuscular junctions, retrograde tracing, and electromyography. These results provide direct evidence that peripheral nerve suture and/or treatment of NOS inhibitors can maintain the homeostasis of oxidative stress-related biomarkers, especially nNOS in neuronal cell bodies. These actions may thus facilitate the axonal regeneration. [Copyright &y& Elsevier]

Abstract: Transplantation of olfactory ensheathing glial cells (OEG) may improve the outcome from spinal cord injury. Proof-of-principle studies in primates are desirable and the feasibility and efficacy of using in vitro expanded OEG should be tested. An intermediate step between the validation of rodent studies and human clinical trials is to study expanded primate OEG (POEG) xenografts in immunotolerant rodents. In this study the time course to generate purified POEG was evaluated as well as their survival, effect on damaged axons of the corticospinal and serotonergic systems, tissue sparing, and chronic locomotor recovery following transplantation. Fifty-seven nude rats underwent T9/10 spinal cord transection. Thirty-eight rats received POEG, 19 controls were injected with cell medium, and 10 received lentivirally-GFP-transfected POEG. Histological evaluation was conducted at 6 weeks, 8 weeks, 14 weeks and 23–24 weeks. Of these 57 rats, 18 were studied with 5-HT immunostaining, 16 with BDA anterograde CST labeling, and six were used for transmission electron microscopy. In grafted animals, behavioral recovery, sprouting and limited regeneration of 5-HT fibers, and increased numbers of proximal collateral processes but not regeneration of CST fibers was observed. Grafted animals had less cavitation in the spinal cord stumps than controls. Behavioral recovery peaked at three months and then declined. Five POEG-transplanted animals that had shown behavioral recovery underwent retransection and behavioral scores did not change significantly, suggesting that long tract axonal regeneration did not account for the locomotor improvement. At the ultrastructural level presumptive POEG were found to have direct contacts with astrocytes forming the glia limitans, distinct from those formed by Schwann cells. At 6 weeks GFP expression was detected in cells within the lesion site and within nerve roots but did not match the pattern of Hoechst nuclear labeling. At 3.5 months only GFP-positive debris in macrophages could be detected. Transplanted POEG support behavioral recovery via mechanisms that appear to be independent of long tract regeneration. [Copyright &y& Elsevier]

Abstract: A lesion of a peripheral nerve before a second injury (conditioning lesion, CL), enhances peripheral and central regeneration of dorsal root ganglion (DRG) axons. This effect is mediated by elevated neuronal cAMP. Here we wanted to investigate whether electrical stimulation (ES) of an intact nerve, which has been shown to accelerate peripheral axon outgrowth, is also effective in promoting axon regeneration of injured DRG axons in vitro and of the central DRG axons in vivo and, whether this effect is mediated by elevation of cAMP. For the in vitro assay, the intact sciatic nerve of adult rats was stimulated at 20 Hz for 1 h, 7 days before harvest and primary culture of DRG neurons on a growth permissive substrate. In the in vivo study, the central axons of the lumbosacral DRGs were cut in the Th8 dorsal column, and the sciatic nerve was either cut or left intact, and subjected to 1 h ES at 20 Hz or 200 Hz. In vitro, ES increased neurite outgrowth 4-fold as compared to non-stimulated DRG neurons. In vivo, ES at 20 Hz significantly increased axon outgrowth into the central lesion site as compared to the Sham control. The 20 Hz ES was as effective as the CL in increasing axon outgrowth into the lesion site but not in promoting axonal elongation even though 20 Hz ES increased intracellular cAMP levels in DRG neurons as effectively as the CL. Thus elevation of cAMP may account for the central axonal outgrowth after ES and a CL. [Copyright &y& Elsevier]

Abstract: Axonal regeneration is normally limited after injuries to CNS white matter. Infusion of neurotrophins has been successful in promoting regenerative growth through injured white matter but this growth generally fails to extend beyond the infusion site. These observations are consistent with a chemotropic effect of these factors on axonal growth and support the prevailing view that neurotrophin-induced axonal regeneration requires the use of gradients, i.e., gradually increasing neurotrophin levels along the target fiber tract. To examine the potential of global overexpression of neurotrophins to promote, and/or modify the orientation of, regenerative axonal growth within white matter, we grafted nerve growth factor (NGF) responsive neurons into the corpus callosum of transgenic mice overexpressing NGF throughout the CNS under control of the promoter for glial fibrillary acidic protein. One week later, glial fibrillary acidic protein and chondroitin sulfate proteoglycan immunoreactivity increased within injured white matter around the grafts. NGF levels were significantly higher in the brains of transgenic compared with non-transgenic mice and further elevated within injury sites compared with the homotypic region of the non-injured side. Although there was minimal outgrowth from neurons grafted into non-transgenic mice, extensive parallel axonal regeneration had occurred within the corpus callosum up to 1.5 mm beyond the astrogliotic scar (the site of maximum NGF expression) in transgenic mice. These results demonstrate that global overexpression of neurotrophins does not override the constraints limiting regenerative growth to parallel orientations and suggest that such factors need not be presented as positive gradients to promote axonal regeneration within white matter. [Copyright &y& Elsevier]

Abstract: Axonal regeneration within the injured central nervous system (CNS) is hampered by multiple inhibitory molecules in the glial scar and the surrounding disrupted myelin. Many of these inhibitors stimulate, either directly or indirectly, the Rho intracellular signaling pathway, providing a strong rationale to target it following spinal cord injuries. In this study, we infused either control (PBS) or a ROCK inhibitor, Y27632 (2 mM or 20 mM, 12 μl/day for 14 days) into the intrathecal space of adult rats starting immediately after a cervical 4/5 dorsal column transection. Histological analysis revealed that high dose-treated animals displayed significantly more axon sprouts in the grey matter distal to injury compared to low dose-treated rats. Only the high dose regimen stimulated sprouting of the dorsal ascending axons along the walls of the lesion cavity. Footprint analysis revealed that the increased base of support normalized significantly faster in control and high dose-treated animals compared to low dose animals. Forepaw rotation angle, and the number of footslips on a horizontal ladder improved significantly more by 6 weeks in high dose animals compared to the other two groups. In a food pellet reaching test, high dose animals performed significantly better than low dose animals, which failed to recover. There was no evidence of mechanical allodynia in any treatment group; however, the slightly shortened heat withdrawal times normalized only with the high dose treatment. Collectively, our data support beneficial effects of high dose Y27632 treatment but indicate that low doses might be detrimental. [Copyright &y& Elsevier]

Abstract: In mature mammals, retinal ganglion cells (RGCs) are generally unable to regenerate injured axons into the optic nerve. Here, we report that an intravitreal injection of either of two macrophage activators, oxidized galectin-1 or zymosan, strongly enhanced the regeneration of transected RGC axons beyond an optic nerve crush site in adult cats. Using WGA-HRP as an anterograde tracer, we found that injection of either macrophage activator caused many axons to grow into the distal optic nerve when evaluated 14 days later, with the strongest effects seen after injecting 100 ng of galectin-1. Elongation continued for at least another 2 weeks. Control eyes injected with saline contained very few labeled axons extending across the crush site. Elevation of intracellular cAMP levels using forskolin also enhanced regeneration beyond the crush site to some extent, but this treatment did not augment the effect of galectin-1 any further. These results indicate that RGCs of adult cats are capable of reverting to an active growth state and at least partially overcoming an inhibitory CNS environment as a result of intravitreal macrophage activation. [Copyright &y& Elsevier]

Abstract: In order to determine the effect of nerve root replantation on motoneuron survival and regeneration, we have avulsed and replanted C7 ventral rootlets in adult rabbits under various conditions. Intraspinal alterations and exact positions of ventrolateral replantations were studied in each animal, and the effects of BDNF and/or CNTF administration during replantation investigated in different experimental groups. Six months after lesion, about 70% of motoneurons were lost on the lesioned sides in the C7 segment, without significant differences between groups. Retrograde fluorescent tracing and histological analysis documented that many axons had regrown through the original ventral exit zones or had exited the spinal cord at the lateral replantation site. However, many laterally exiting axons had not grown out directly from the ventral horn through the lateral white matter but had elongated vertically before leaving the spinal cord. The mean axonal diameter was significantly higher in regenerated axons that had exited through the original ventral exit zones in comparison with axons which had grown out laterally. Application of BDNF and/or CNTF did not show any effects on the pathways of regeneration into the replanted root. The results indicate that motoneuron survival cannot be significantly improved by a single dose of neurotrophic factors applied to a ventrolateral replantation site. However, a significant number of myelinating axons are found in replanted roots, and regeneration may be more efficient when outgrowth through the original ventral exit zone is supported. [Copyright &y& Elsevier]

Abstract: Previously, our lab reported the isolation of patient-specific neurosphere-forming progenitor lines from human adult olfactory epithelium from cadavers as well as patients undergoing nasal sinus surgery. RT-PCR and ELISA demonstrated that the neurosphere-forming cells (NSFCs) produced BDNF. Since rubrospinal tract (RST) neurons have been shown to respond to exogenous BNDF, it was hypothesized that if the NSFCs remained viable following engraftment into traumatized spinal cord, they would rescue axotomized RS neurons from retrograde cell atrophy and promote functional recovery. One week after a partial cervical hemisection, GFP-labeled NSFCs suspended in Matrigel® matrix or Matrigel® matrix alone was injected into the lesion site. GFP-labeled cells survived up to 12 weeks in the lesion cavity or migrated within the ipsilateral white matter; the apparent number and mean somal area of fluorogold (FG)-labeled axotomized RST neurons were greater in the NSFC-engrafted rats than in lesion controls. Twelve weeks after engraftment, retrograde tracing with FG revealed that some RST neurons regenerated axons 4–5 segments caudal to the engraftment site; anterograde tracing with biotinylated dextran amine confirmed regeneration of RST axons through the transplants within the white matter for 3–6 segments caudal to the grafts. A few RST axons terminated in gray matter close to motoneurons. Matrix alone did not elicit regeneration. Behavioral analysis revealed that NSFC-engrafted rats displayed better performance during spontaneous vertical exploration and horizontal rope walking than lesion Matrigel® only controls 11 weeks post transplantation. These results emphasize the unique potential of human olfactory neuroepithelial-derived progenitors as an autologous source of stem cells for spinal cord repair. [Copyright &y& Elsevier]

Abstract: The expression of the transcription factor ATF3 in the brain was examined by immunohistochemistry during axonal regeneration induced by the implantation of pieces of peripheral nerve into the thalamus of adult rats. After 3 days, ATF3 immunoreactivity was present in many cells within approximately 500 μm of the graft. In addition, ATF3-positive cell nuclei were found in the thalamic reticular nucleus (TRN) and medial geniculate nuclear complex (MGN), from which most regenerating axons originate. CNS cells with ATF3-positive nuclei were predominantly neurons and did not show signs of apoptosis. The number of ATF3-positive cells had declined by 7 days and further by 1 month after grafting when most ATF3-positive cells were found in the TRN and MGN. 14 days or more after grafting, some ATF3-positive nuclei were distorted and may have been apoptotic. In some experiments of 1 month duration, neurons which had regenerated axons to the distal ends of grafts were retrogradely labeled with DiAsp. ATF3-positive neurons in these animals were located in regions of the TRN and MGN containing retrogradely labeled neurons and the great majority were also labeled with DiAsp. SCG10 and c-Jun were found in neurons in the same regions as retrogradely labeled and ATF3-positive cells. Thus, ATF3 is transiently upregulated by injured CNS neurons, but prolonged expression is part of the pattern of gene expression associated with axonal regeneration. The co-expression of ATF3 with c-jun suggests that interactions between these transcription factors may be important for controlling the program of gene expression necessary for regeneration. [Copyright &y& Elsevier]

The regeneration of sciatic-dorsal column (DC) axons following DC crush injury and treatment with olfactory ensheathing cells (OECs) and/or sciatic axotomy (“conditioning lesion”) was evaluated. Sciatic-DC axons were examined with a transganglionic tracer, cholera toxin conjugated to horseradish peroxidase, and evaluated at chronic time points, 2–26 weeks post-lesion. With DC injury alone (n = 7), sciatic-DC axons were localized to the caudal border of the lesion terminating in reactive end bulbs with no indication of growth into the lesion. In contrast, treatment with either a heterogeneous population of OECs (equal numbers of p75- and fibronectin-positive OECs) (n = 9) or an enriched population of OECs (75% p75-positive OECs) (n = 6) injected either directly into the lesion or 1-mm rostral and caudal to the injury, stimulated DC axon growth into the lesion. A similar regenerative response was observed with a conditioning lesion either concurrent to (n = 4) or 1 week before (n = 4) the DC injury. In either of the latter two paradigms, some DC axons grew across the injury, but no axons grew into the rostral intact spinal cord. Upon combining OEC treatment with the conditioning lesion (n = 21), the result was additive, increasing DC axon growth beyond the rostral border of the lesion in best cases. Additional factors that may limit DC regeneration were tested including formation of the glial scar (immunoreactivity to glial fibrillary acidic protein in astrocytes and to chondroitin sulfate proteoglycans), which remained similar between treated and untreated groups. [Copyright &y& Elsevier]

The repair of axonal projections and the reconstruction of neuronal circuits after CNS lesions or during neurodegenerative disease are major challenges in restorative neuroscience. We have explored the potential of transplanted immature neurons to repair a specific axonal projection in an entorhino-hippocampal slice culture model system. When slices of immature entorhinal cortex (EC) from tau-GFP transgenic mice were cultured next to slices from postnatal hippocampus, an axonal projection from the E18 embryonic entorhinal cortex to the dentate gyrus of the postnatal hippocampus developed, which was similar to that observed in control cultures. Even more immature neuronal precursors in slices from E15 developing cerebral cortex differentiated and established an axonal projection to the hippocampal slice. This projection terminated specifically in the outer molecular layer of the dentate gyrus, the normal target area of the entorhino-hippocampal projection. When embryonic tissue from the presumptive brainstem area was used, there was still a subpopulation of fibers with a specific termination in the outer molecular layer, but few specific fibers were found in cocultures with embryonic midbrain. Our results show that very immature cortical neurons are potentially able to form an entorhino-hippocampal projection that terminates in a correct lamina-specific fashion in the dentate gyrus. These findings support the idea that immature neuronal precursor cells could be used for the reconstruction of specific neuronal circuits. [Copyright &y& Elsevier]

Tumor necrosis factor alpha (TNF)-induced cellular signaling through the p38 mitogen-activated protein kinase (p38 MAPK) pathway plays a critical role in Wallerian degeneration and subsequent regeneration, processes that depend on Schwann cell (SC) activity. TNF dose-dependently induces Schwann cell and macrophage activation in vivo and apoptosis in primary SC cultures in vitro, while inhibition of p38 MAPK is thought to block these cellular processes. We show with Western blots that after sciatic nerve crush injury, phosphorylated p38 (p-p38) MAPK is significantly increased (P < 0.01) in distal nerve segments. In tissue sections, p38 co-localized immunohistochemically with activated Schwann cells (GFAP) and to a lesser degree with macrophages (ED-1). In other experiments, animals were gavaged with Scios SD-169 (10 or 30 mg/kg) or excipient (PEG300) 1 day before and daily after crush injury to the sciatic nerve. SD-169 is a proprietary oral inhibitor of p38 MAPK activity. The rate of axonal regeneration was determined by the functional pinch test and was significantly increased in treated animals 8 days after crush injury (P < 0.05; 30 mg/kg dose). In SD-169-treated animals with nerve transection, nerve fibers regenerating through a silicone chamber were morphologically more mature than untreated nerves when observed 28 days after transection. TNF immunofluorescence of distal nerve segments after crush injury suggested that SD-169 reduced SC TNF protein. In support of these findings, SD-169 significantly reduced (P < 0.05) TNF-mediated primary SC death in culture experiments. We conclude that inhibition of p38 activity promotes axonal regeneration through interactions with SC signaling and TNF activity. [Copyright &y& Elsevier]

Glial cell line-derived neurotrophic factor (GDNF), a distant member of the transforming growth factor-β (TGF-β) family, is widely expressed in the developing and adult central nervous system (CNS). At present, limited information is available regarding the effects of GDNF in the repair of spinal cord injury (SCI). In the present study, mini-guidance channels containing either: (1) Matrigel (MG, a basement membrane component), (2) Schwann cells (SCs, 120 × 106/ml) in MG (SC-MG), (3) recombinant human GDNF (rhGDNF, 3 μg/μl) in MG (GDNF-MG), and (4) a combination of all three components (GDNF-SC-MG) were grafted into a T9 hemisection-gap lesion in adult rats to examine the effects of GDNF on axonal regeneration and myelination following SCI. Thirty days post-transplantation, limited axonal growth was observed within guidance channels containing MG-alone (MG). When SCs were added to the channels (SC-MG group), consistent axonal ingrowth containing both myelinated and unmyelinated axons was observed, confirming our previous findings. The addition of GDNF-alone without SCs (GDNF-MG) resulted in substantial ingrowth of unmyelinated axons, suggesting that GDNF has a direct neurite-growth promoting effect on these axons. Implantation of channels containing both GDNF and SCs (GDNF-SC-MG) produced a significant and synergistic increase in axonal regeneration and myelination. In addition, GDNF reduced the extent of reactive gliosis, infiltration of activated macrophages/microglia, and cystic cavitation at the graft-host interfaces. Retrograde tracing revealed that grafts of SC-seeded channels containing GDNF promoted a significant increase in the number of propriospinal neurons which had regenerated their axons into the grafts, as compared to SC-MG-seeded channels. These results indicate that GDNF may play a novel therapeutic role in promoting propriospinal axonal regeneration, enhancing myelin formation, and improving graft-host interfaces after SCI. [Copyright &y& Elsevier]

We have examined the expression of the immunophilin FKBP12 in adult rat intrinsic CNS neurons stimulated to regenerate axons by the implantation of segments of autologous tibial nerve into the thalamus or cerebellum. After survival times of 3 days to 6 weeks, the brains were fresh-frozen. In some animals the regenerating neurons were retrogradely labelled with cholera toxin subunit B 1 day before they were killed. Sections through the thalamus or cerebellum were used for in situ hybridization with digoxygenin-labelled riboprobes for FKBP12 or immunohistochemistry to detect cholera toxin subunit B-labelled neurons. FKBP12 was constitutively expressed by many neurons, and was very strongly expressed in the hippocampus and by Purkinje cells. Regenerating neurons were found in the thalamic reticular nucleus and deep cerebellar nuclei of animals that received living grafts. Neurons in these nuclei upregulated FKBP12 mRNA; such neurons were most numerous at 3 days post grafting but were most strongly labelled at 2 weeks post grafting. Regenerating neurons identified by retrograde labelling were found to have upregulated FKBP12 mRNA. No upregulation was seen in neurons in animals that received freeze-killed grafts, which do not support axonal regeneration. We conclude that FKBP12 is a regeneration-associated gene in intrinsic CNS neurons. [Copyright &y& Elsevier]

Olfactory ensheathing cells (OEC) constitute a specialized population of glia that accompany primary olfactory axons and have been reported to facilitate axonal regeneration after spinal cord injury in vivo. In the present report we describe OEC neurotrophic factor expression and neurotrophic properties of OECs in vitro. Investigation of the rat olfactory system during development and adulthood by radioactive in situ hybridization revealed positive labeling in the olfactory nerve layer for the neurotrophic molecules S-100β, CNTF, BMP-7/OP-1, and artemin, as well as for the neurotrophic factor receptors RET and TrkC. Ribonuclease protection assay of cultured OEC revealed expression of NGF, BDNF, GDNF, and CNTF mRNA, while NT3 and NT4 mRNA were not detectable. In vitro bioassays of neurotrophic activity involved coculturing of adult OEC with embryonic chick ganglia and demonstrated increased neurite outgrowth from sympathetic, ciliary, and Remak’s ganglia. However, when culturing the ganglia with OEC-conditioned medium, neurite outgrowth was not stimulated to any detectable extent. Our results suggest that the neurotrophic properties of OEC may involve secretion of neurotrophic molecules but that cellular interactions are crucial. [Copyright &y& Elsevier]

Neurogenic differentiation 1 (NeuroD1) is mainlyexpressed in developing neurons where it plays critical roles in neuronal maturation and neurite elongation. The potential role and mechanism of NeuroD1 in adult axonal regeneration is not clear. The present study used synapsin (SYN) Cre and AAV9-Flex vectors to conditionally overexpress NeuroD1 in adult spinal neurons and found that NeuroD1 overexpression significantly accelerated axonal regeneration and functional recovery after sciatic nerve injury. Further in vitro and in vivo experiments suggested that the mechanism of NeuroD1 promotion on axonal regeneration was related to its regulation of the expression of neurotrophin BDNF and its receptor TrkB as well as a microtubule severing protein spastin. • NeuroD1 overexpression facilitates axonal regeneration after peripheral nerve injury. • NeuroD1 overexpression reverses the functional impairment of the injured nerve. • NeuroD1 overexpression upregulates the expression of BDNF, TrkB and Spastin. [ABSTRACT FROM AUTHOR]

The adult mammalian central nervous system (CNS) rarely recovers from injury. Myelin fragments contain axonal growth inhibitors that limit axonal regeneration, thus playing a major role in determining neural recovery. Nogo receptor-1 (NgR1) and its ligands are among the inhibitors that limit axonal regeneration. It has been previously shown that the endogenous protein, lateral olfactory tract usher substance (LOTUS), antagonizes NgR1-mediated signaling and accelerates neuronal plasticity after spinal cord injury and cerebral ischemia in mice. However, it remained unclear whether LOTUS-mediated reorganization of descending motor pathways in the adult brain is physiologically functional and contributes to functional recovery. Here, we generated LOTUS-overexpressing transgenic (LOTUS-Tg) rats to investigate the role of LOTUS in neuronal function after damage. After unilateral pyramidotomy, motor function in LOTUS-Tg rats recovered significantly compared to that in wild-type animals. In a retrograde tracing study, labeled axons spanning from the impaired side of the cervical spinal cord to the unlesioned hemisphere of the red nucleus and sensorimotor cortex were increased in LOTUS-Tg rats. Anterograde tracing from the unlesioned cortex also revealed enhanced ipsilateral connectivity to the impaired side of the cervical spinal cord in LOTUS-Tg rats. Moreover, electrophysiological analysis showed that contralesional cortex stimulation significantly increased ipsilateral forelimb movement in LOTUS-Tg rats, which was consistent with the histological findings. According to these data, LOTUS overexpression accelerates ipsilateral projection from the unlesioned cortex and promotes functional recovery after unilateral pyramidotomy. LOTUS could be a future therapeutic option for CNS injury. • LOTUS-Tg rats allowed the study of endogenous NgR1 antagonist function. • LOTUS was overexpressed in the cervical spinal cord after unilateral pyramidotomy. • LOTUS-Tg rats had faster functional improvement after unilateral pyramidotomy. • LOTUS promoted neural plasticity and unlesioned cortex–denervated side connection. • LOTUS promoted functional improvement after central nervous system injury. [ABSTRACT FROM AUTHOR]