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The Journal of Neuroscience : the... Sep 2012Previous studies have shown that injured dorsal column sensory axons extend across a spinal cord lesion site if axons are guided by a gradient of neurotrophin-3 (NT-3)...
Previous studies have shown that injured dorsal column sensory axons extend across a spinal cord lesion site if axons are guided by a gradient of neurotrophin-3 (NT-3) rostral to the lesion. Here we examined whether continuous NT-3 delivery is necessary to sustain regenerated axons in the injured spinal cord. Using tetracycline-regulated (tet-off) lentiviral gene delivery, NT-3 expression was tightly controlled by doxycycline administration. To examine axon growth responses to regulated NT-3 expression, adult rats underwent a C3 dorsal funiculus lesion. The lesion site was filled with bone marrow stromal cells, tet-off-NT-3 virus was injected rostral to the lesion site, and the intrinsic growth capacity of sensory neurons was activated by a conditioning lesion. When NT-3 gene expression was turned on, cholera toxin β-subunit-labeled sensory axons regenerated into and beyond the lesion/graft site. Surprisingly, the number of regenerated axons significantly declined when NT-3 expression was turned off, whereas continued NT-3 expression sustained regenerated axons. Quantification of axon numbers beyond the lesion demonstrated a significant decline of axon growth in animals with transient NT-3 expression, only some axons that had regenerated over longer distance were sustained. Regenerated axons were located in white matter and did not form axodendritic synapses but expressed presynaptic markers when closely associated with NG2-labeled cells. A decline in axon density was also observed within cellular grafts after NT-3 expression was turned off possibly via reduction in L1 and laminin expression in Schwann cells. Thus, multiple mechanisms underlie the inability of transient NT-3 expression to fully sustain regenerated sensory axons.
Topics: Analysis of Variance; Animals; Antigens; Axons; Cell Transplantation; Cells, Cultured; Cholera Toxin; Disease Models, Animal; Doxycycline; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Genetic Therapy; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; HEK293 Cells; Humans; Laminin; Leukocyte L1 Antigen Complex; Membrane Glycoproteins; Microtubule-Associated Proteins; Myelin-Oligodendrocyte Glycoprotein; Nerve Growth Factors; Nerve Regeneration; Nerve Tissue Proteins; Neurofilament Proteins; Neurotrophin 3; Proteoglycans; Rats; Rats, Inbred F344; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Schwann Cells; Sciatic Nerve; Sensory Receptor Cells; Spinal Cord Injuries; Stem Cell Transplantation; Time Factors; Transfection
PubMed: 22993437
DOI: 10.1523/JNEUROSCI.5041-11.2012 -
Journal of Neuroscience Methods Nov 2012Techniques used to produce partial spinal cord injuries in animal models have the potential for creating variability in lesions. The amount of tissue affected may...
Techniques used to produce partial spinal cord injuries in animal models have the potential for creating variability in lesions. The amount of tissue affected may influence the functional outcomes assessed in the animals. The recording of somatosensory evoked potentials (SSEPs) may be a valuable tool for assessing the extent of lesion applied in animal models of traumatic spinal cord injury (SCI). Intraoperative tibial SSEP recordings were assessed during surgically induced lateral thoracic hemisection SCI in Sprague-Dawley rats. The transmission of SSEPs, or lack thereof, was determined and compared against the integrity of the dorsal funiculi on each side of the spinal cord upon histological sectioning. An association was found between the presence of an SSEP signal and presence of intact dorsal funiculus tissue. The relative risk is 4.50 (95% confidence interval: 1.83-11.08) for having an intact dorsal funiculus when the ipsilateral SSEP was present compared to when it was absent. Additionally, the amount of spared spinal cord tissue correlates with final functional assessments at nine weeks post injury: BBB (linear regression, R²=0.618, p<0.001) and treadmill test (linear regression, R²=0.369, p=0.016). Therefore, we propose intraoperative SSEP monitoring as a valuable tool to assess extent of lesion and reduce variability between animals in experimental studies of SCI.
Topics: Animals; Disease Models, Animal; Evoked Potentials, Somatosensory; Female; Monitoring, Intraoperative; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries
PubMed: 22960163
DOI: 10.1016/j.jneumeth.2012.08.024 -
Neuroscience Letters Sep 2012In a previous study we found that the EphA4 receptor inhibits regeneration following spinal cord injury by blocking regrowth of axons and regulation of astrocyte...
In a previous study we found that the EphA4 receptor inhibits regeneration following spinal cord injury by blocking regrowth of axons and regulation of astrocyte reactivity. In our original studies using EphA4 null mice [Goldshmit et al., J. Neurosci., 2004] we found attenuated astrocyte reactivity following spinal cord injury. Several other studies have now supported the role of EphA4 in regulating neural regeneration but a recent study [Herrmann et al., Exp. Neurol., 2010] did not find an effect of EphA4 on astrocyte reactivity. Re-examination of astrocytic gliosis following injury in our current cohort of EphA4 null mice revealed that they no longer showed attenuation of astrocyte reactivity, however other EphA4 null mouse phenotypes, such as decreased size of the dorsal funiculus were unaltered. We hypothesised that long-term breeding on the C57Bl/6 background may influence the EphA4-mediated astrocyte phenotype and compared astrocytic gliosis at 4 days following spinal cord injury in wildtype and EphA4 null mice on the C57Bl/6 background and backcrossed C57Bl/6×129Sv(F2) mice, as well as wildtype 129Sv mice. 129Sv mice had increased GFAP expression and increased numbers of reactive GFAP astrocytes compared to C57Bl/6 mice. There was no significant effect of EphA4 deletion on GFAP expression in C57Bl/6 mice or the F2 crosses other than a moderately decreased number of EphA4 null astrocytes in C57Bl/6 mice using one of two antibodies. Therefore, there has been an apparent change in EphA4-mediated astroglial phenotype associated with long term breeding of the EphA4 colony but it does not appear to be influenced by background mouse strain.
Topics: Animals; Astrocytes; Breeding; Cell Proliferation; Female; Glial Fibrillary Acidic Protein; Gliosis; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Receptor, EphA4; Species Specificity; Spinal Cord Injuries; Up-Regulation
PubMed: 22824304
DOI: 10.1016/j.neulet.2012.07.023 -
The Journal of Neuroscience : the... Apr 2012Hindbrain dorsal interneurons that comprise the rhombic lip relay sensory information and coordinate motor outputs. The progenitor dA1 subgroup of interneurons, which is...
Hindbrain dorsal interneurons that comprise the rhombic lip relay sensory information and coordinate motor outputs. The progenitor dA1 subgroup of interneurons, which is formed along the dorsal-most region of the caudal rhombic lip, gives rise to the cochlear and precerebellar nuclei. These centers project sensory inputs toward upper-brain regions. The fundamental role of dA1 interneurons in the assembly and function of these brainstem nuclei is well characterized. However, the precise en route axonal patterns and synaptic targets of dA1 interneurons are not clear as of yet. Novel genetic tools were used to label dA1 neurons and trace their axonal trajectories and synaptic connections at various stages of chick embryos. Using dA1-specific enhancers, two contralateral ascending axonal projection patterns were identified; one derived from rhombomeres 6-7 that elongated in the dorsal funiculus, while the other originated from rhombomeres 2-5 and extended in the lateral funiculus. Targets of dA1 axons were followed at later stages using PiggyBac-mediated DNA transposition. dA1 axons were found to project and form synapses in the auditory nuclei and cerebellum. Investigation of mechanisms that regulate the patterns of dA1 axons revealed a fundamental role of Lim-homeodomain (HD) proteins. Switch in the expression of the specific dA1 Lim-HD proteins Lhx2/9 into Lhx1, which is typically expressed in dB1 interneurons, modified dA1 axonal patterns to project along the routes of dB1 subgroup. Together, the results of this research provided new tools and knowledge to the assembly of trajectories and connectivity of hindbrain dA1 interneurons and of molecular mechanisms that control these patterns.
Topics: Age Factors; Animals; Axons; Basic Helix-Loop-Helix Transcription Factors; Body Patterning; Cell Count; Chick Embryo; Ectodysplasins; Electroporation; Functional Laterality; Gene Expression Regulation, Developmental; Green Fluorescent Proteins; Humans; Interneurons; LIM-Homeodomain Proteins; Nerve Tissue Proteins; PAX2 Transcription Factor; Rhombencephalon; Synapses
PubMed: 22539838
DOI: 10.1523/JNEUROSCI.4231-11.2012 -
Neural Regeneration Research Feb 2012Fluoro-ruby was injected into the posterior funiculus of the spinal cord in the cervical (C5-T2) and lumbar (L3-6) segments of adult guinea pigs. The spinal cord was cut...
Fluoro-ruby was injected into the posterior funiculus of the spinal cord in the cervical (C5-T2) and lumbar (L3-6) segments of adult guinea pigs. The spinal cord was cut into serial frozen sections. The Fluoro-ruby labeling was clearly delineated from the surrounding structure. The labeling traversed the cervical, thoracic and lumbar segments, and was located on the ventral portion of the posterior funiculus on the injected side, proximal to the intermediate zone of the dorsal gray matter. The fluorescence area narrowed rostro-caudally. The spinal cord, spinal cord gray matter and corticospinal tract were reconstructed using 3D-DOCTOR 4.0 software, resulting in a robust three-dimensional profile. Using functionality provided by the reconstruction software, free multi-angle observation and sectioning could be conducted on the spinal cord and corticospinal tract. Our experimental findings indicate that the Fluoro-ruby retrograde fluorescent tracing technique can accurately display the anatomical location of corticospinal tract in the guinea pig and that three-dimensional reconstruction software can be used to provide a three-dimensional image of the corticospinal tract.
PubMed: 25774176
DOI: 10.3969/j.issn.1673-5374.2012.05.007 -
Journal of Neurotrauma Feb 2012Currently, few interventions have been shown to successfully limit the progression of secondary damage events associated with the acute phase of spinal cord injury...
Currently, few interventions have been shown to successfully limit the progression of secondary damage events associated with the acute phase of spinal cord injury (SCI). Docosahexaenoic acid (DHA, C22:6 n-3) is neuroprotective when administered following SCI, but its potential as a pretreatment modality has not been addressed. This study used a novel DHA pretreatment experimental paradigm that targets acute cellular and molecular events during the first week after SCI in rats. We found that DHA pretreatment reduced functional deficits during the acute phase of injury, as shown by significant improvements in Basso-Beattie-Bresnahan (BBB) locomotor scores, and the detection of transcranial magnetic motor evoked potentials (tcMMEPs) compared to vehicle-pretreated animals. We demonstrated that, at 7 days post-injury, DHA pretreatment significantly increased the percentage of white matter sparing, and resulted in axonal preservation, compared to the vehicle injections. We found a significant increase in the survival of NG2+, APC+, and NeuN+ cells in the ventrolateral funiculus (VLF), dorsal corticospinal tract (dCST), and ventral horns, respectively. Interestingly, these DHA protective effects were observed despite the lack of inhibition of inflammatory markers for monocytes/macrophages and astrocytes, ED1/OX42 and GFAP, respectively. DHA pretreatment induced levels of Akt and cyclic AMP responsive element binding protein (CREB) mRNA and protein. This study shows for the first time that DHA pretreatment ameliorates functional deficits, and increases tissue sparing and precursor cell survival. Further, our data suggest that DHA-mediated activation of pro-survival/anti-apoptotic pathways may be independent of its anti-inflammatory effects.
Topics: Animals; Cell Death; Cyclic AMP Response Element-Binding Protein; Docosahexaenoic Acids; Evoked Potentials, Motor; Female; Fluorescent Antibody Technique; Hindlimb; In Situ Nick-End Labeling; Inflammation; Injections, Intravenous; Locomotion; Macrophage Activation; Microglia; Myelin Sheath; Neural Conduction; Neural Stem Cells; Neuroprotective Agents; Oncogene Protein v-akt; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Signal Transduction; Spinal Cord Injuries
PubMed: 21970623
DOI: 10.1089/neu.2011.2141 -
PloS One 2011Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we...
Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.
Topics: Animals; Axons; Female; GTPase-Activating Proteins; Immunohistochemistry; Immunoprecipitation; In Situ Hybridization; Mice; Mice, Knockout; Nerve Tissue Proteins; Pregnancy; Protein Binding; Receptors, Immunologic; Spinal Cord; Roundabout Proteins
PubMed: 21655271
DOI: 10.1371/journal.pone.0019887 -
The Journal of Comparative Neurology Oct 2011Over the past century, the question of whether the cells of origin of the corticospinal tract (CST) die following spinal cord injury (SCI) has been debated. A recent...
Over the past century, the question of whether the cells of origin of the corticospinal tract (CST) die following spinal cord injury (SCI) has been debated. A recent study reported an approximately 20% loss of retrogradely labeled cortical motoneurons following damage to their axons resulting from SCI at T9 (Hains et al. [2003] J. Comp. Neurol. 462:328-341). In follow-up studies, however, we failed to find any evidence of loss of CST axons in the medullary pyramid, which must occur if CST neurons die. Here, we seek to resolve the discrepancy by re-evaluating possible loss of CST neurons using the same techniques as Hains et al. (quantitative analysis of retrograde labeling and staining for cell death markers including TUNEL and Hoechst labeling of the nuclei). Following either dorsal funiculus lesions at thoracic level 9 (T9) or lateral hemisection at cervical level 5 (C5), our results reveal no evidence for a loss of retrogradely labeled neurons and no evidence for TUNEL staining of axotomized cortical motoneurons. These results indicate that CST cell bodies do not undergo retrograde cell death following SCI, and therefore targeting such cell death is not a valid therapeutic target.
Topics: Animals; Apoptosis; Cerebral Cortex; Female; In Situ Nick-End Labeling; Motor Neurons; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries
PubMed: 21618218
DOI: 10.1002/cne.22661 -
Frontiers in Neuroengineering 2011By electrically stimulating the spinal cord, it is possible to activate functional populations of neurons that modulate motor and sensory function. One method for...
By electrically stimulating the spinal cord, it is possible to activate functional populations of neurons that modulate motor and sensory function. One method for accessing these neurons is via their associated axons, which project as functionally segregated longitudinal columns of white-matter funiculi (i.e., spinal tracts). To stimulate spinal tracts without penetrating the cord, we have recently developed technology that enables close-proximity, multi-electrode contact with the spinal cord surface. Our stretchable microelectrode arrays (sMEAs) are fabricated using an elastomer polydimethylsiloxane substrate and can be wrapped circumferentially around the spinal cord to optimize electrode contact. Here, sMEAs were used to stimulate the surfaces of rat spinal cords maintained in vitro, and their ability to selectively activate axonal surface tracts was compared to rigid bipolar tungsten microelectrodes pressed firmly onto the cord surface. Along dorsal column tracts, the axonal response to sMEA stimulation was compared to that evoked by rigid microelectrodes through measurement of their evoked axonal compound action potentials (CAPs). Paired t-tests failed to reveal significant differences between the sMEA's and the rigid microelectrode's stimulus resolution, or in their ranges of evoked CAP conduction velocities. Additionally, dual-site stimulation using sMEA electrodes recruited spatially distinct populations of spinal axons. Site-specific stimulation of the ventrolateral funiculus - a tract capable of evoking locomotor-like activity - recruited ventral root efferent activity that spanned several spinal segments. These findings indicate that the sMEA stimulates the spinal cord surface with selectivity similar to that of rigid microelectrodes, while possessing potential advantages concerning circumferential contact and mechanical compatibility with the cord surface.
PubMed: 21541256
DOI: 10.3389/fneng.2011.00005 -
Anatomical Record (Hoboken, N.J. : 2007) May 2011Amyotrophic lateral sclerosis (ALS) is a progressively fatal, incurable, neurodegenerative disorder. In this study, we investigated whether olfactory ensheathing cells...
Amyotrophic lateral sclerosis (ALS) is a progressively fatal, incurable, neurodegenerative disorder. In this study, we investigated whether olfactory ensheathing cells (OEC) transplantation could provide protection to motor neurons and enable remyelination in mutant SOD1(G93A) transgenic rats with ALS. Seventy-two rats were divided into four groups: SOD1(G93A) rats (n = 20); medium+SOD1(G93A) rats (n = 20); OECs+SOD1(G93A) rats (n = 24); and another eight wild-type rats were used as controls. About 5 μL (1 × 10(5)) OECs in DF12 medium was injected into the dorsal funiculus of the thoracic spinal cord at a predetermined depth. Survival analysis revealed a significant increase in the survival time in OEC+SOD1(G93A) rats. Body weight records and inclined board test showed a significant difference between OEC+SOD1(G93A) and SOD1(G93A) from the onset at 7 days to 11 days (P < 0.05). Four weeks following transplantation, motor neuron counts in the ventral horn of the spinal cord noted a significant motor neuron loss in SOD1(G93A) rats when compared with wild-type rats (P < 0.001), and much less neuronal loss and collapse was noted in OEC+SOD1(G93A) rats when compared with SOD1(G93A) rats(P < 0.001); immunohistochemistry and Western blot analysis of choline acetyltransferase supported the motor neuron count. Images of confocal microscope indicated that the transplanted OECs had survived for more than 4 weeks and migrated 4.2 mm through the spinal cord. Evidence of remyelination of transplanted OEC was captured with triple fluorescence labeling of green fluorescent protein, neurofilament, and myelin basic protein and was further confirmed by Western blot analysis of MPB. In conclusion, the transplanted OECs could serve as a source of neuroprotection and remyelination to modify the ALS microenvironment.
Topics: Amyotrophic Lateral Sclerosis; Animals; Animals, Genetically Modified; Demyelinating Diseases; Disease Models, Animal; Male; Motor Neurons; Mutation; Nerve Degeneration; Neuroprotective Agents; Olfactory Bulb; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Superoxide Dismutase; Superoxide Dismutase-1; Survival Rate
PubMed: 21416627
DOI: 10.1002/ar.21362