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The Journal of Neuroscience : the... Apr 2000Axonal injury is a feature of traumatic brain injury (TBI) contributing to both morbidity and mortality. The traumatic axon injury (TAI) results from focal perturbations...
Axonal injury is a feature of traumatic brain injury (TBI) contributing to both morbidity and mortality. The traumatic axon injury (TAI) results from focal perturbations of the axolemma, allowing for calcium influx triggering local intraaxonal cytoskeletal and mitochondrial damage. This mitochondrial damage has been posited to cause local bioenergetic failure, leading to axonal failure and disconnection; however, this mitochondrial damage may also lead to the release of cytochrome c (cyto-c), which then activates caspases with significant adverse intraaxonal consequences. In the current communication, we examine this possibility. Rats were subjected to TBI, perfused with aldehydes at 15-360 min after injury, and processed for light microscopic (LM) and electron microscopic (EM) single-labeling immunohistochemistry to detect extramitochondrially localized cytochrome c (cyto-c) and the signature protein of caspase-3 activation (120 kDa breakdown product of alpha-spectrin) in TAI. Combinations of double-labeling fluorescent immunohistochemistry (D-FIHC) were also used to demonstrate colocalization of calpain activation with cyto-c release and caspase-3-induction. In foci of TAI qualitative-quantitative LM demonstrated a parallel, significant increase in cyto-c release and caspase-3 activation over time after injury. EM analysis demonstrated that cyto-c and caspase-3 immunoreactivity were associated with mitochondrial swelling-disruption in sites of TAI. Furthermore, D-IFHC revealed a colocalization of calpain activation, cyto-c release, and caspase-3 induction in these foci, which also revealed progressive TAI. The results demonstrate that cyto-c and caspase-3 participate in the terminal processes of TAI. This suggests that those factors that play a role in the apoptosis in the neuronal soma are also major contributors to the demise of the axonal appendage.
Topics: Animals; Brain Injuries; Calpain; Caspase 3; Caspases; Cytochrome c Group; Diffuse Axonal Injury; Enzyme Activation; Rats; Rats, Sprague-Dawley
PubMed: 10751434
DOI: 10.1523/JNEUROSCI.20-08-02825.2000 -
Brazilian Journal of Medical and... Dec 2000In this study we describe the early changes of the myelin sheath following surgical nerve crush. We used the freeze-fracture technique to better evaluate myelin...
In this study we describe the early changes of the myelin sheath following surgical nerve crush. We used the freeze-fracture technique to better evaluate myelin alterations during an early stage of Wallerian degeneration. Rat sural nerves were experimentally crushed and animals were sacrificed by transcardiac perfusion 30 h after surgery. Segments of the nerves were processed for routine transmission electron microscopy and freeze-fracture techniques. Our results show that 30 h after the lesion there was asynchrony in the pattern of Wallerian degeneration, with different nerve fibers exhibiting variable degrees of axon disruption. This was observed by both techniques. Careful examination of several replicas revealed early changes in myelin membranes represented by vacuolization and splitting of consecutive lamellae, rearrangement of intramembranous particles and disappearance of paranodal transverse bands associated or not with retraction of paranodal myelin terminal loops from the axolemma. These alterations are compatible with a direct injury to the myelin sheath following nerve crush. The results are discussed in terms of a similar mechanism underlying both axon and myelin breakdown.
Topics: Animals; Freeze Fracturing; Microscopy, Electron; Myelin Sheath; Nerve Crush; Rats; Rats, Wistar; Sural Nerve; Wallerian Degeneration
PubMed: 11105101
DOI: 10.1590/s0100-879x2000001200012 -
Biophysical Journal Oct 2000After axonal severance, a barrier forms at the cut ends to rapidly restrict bulk inflow and outflow. In severed crayfish axons we used the exclusion of hydrophilic,...
After axonal severance, a barrier forms at the cut ends to rapidly restrict bulk inflow and outflow. In severed crayfish axons we used the exclusion of hydrophilic, fluorescent dye molecules of different sizes (0.6-70 kDa) and the temporal decline of ionic injury current to levels in intact axons to determine the time course (0-120 min posttransection) of barrier formation and the posttransection time at which an axolemmal ionic seal had formed, as confirmed by the recovery of resting and action potentials. Confocal images showed that the posttransection time of dye exclusion was inversely related to dye molecular size. A barrier to the smallest dye molecule formed more rapidly (<60 min) than did the barrier to ionic entry (>60 min). These data show that axolemmal sealing lacks abrupt, large changes in barrier permeability that would be expected if a seal were to form suddenly, as previously assumed. Rather, these data suggest that a barrier forms gradually and slowly by restricting the movement of molecules of progressively smaller size amid injury-induced vesicles that accumulate, interact, and form junctional complexes with each other and the axolemma at the cut end. This process eventually culminates in an axolemmal ionic seal, and is not complete until ionic injury current returns to baseline levels measured in an undamaged axon.
Topics: Animals; Astacoidea; Axons; Biophysical Phenomena; Biophysics; Fluorescent Dyes; In Vitro Techniques; Ion Channels; Microscopy, Confocal; Nerve Regeneration; Permeability
PubMed: 11023894
DOI: 10.1016/S0006-3495(00)76438-1 -
Journal of Neurochemistry Aug 2000Microtubule-associated protein (MAP) 1B is a high-molecular-weight cytoskeletal protein that is abundant in developing neuronal processes and appears to be necessary for...
Microtubule-associated protein (MAP) 1B is a high-molecular-weight cytoskeletal protein that is abundant in developing neuronal processes and appears to be necessary for axonal growth. Various biochemical and immunocytochemical results are reported, indicating that a significant fraction of MAP1B is expressed as an integral membrane glycoprotein in vesicles and the plasma membrane of neurons. MAP1B is present in microsomal fractions isolated from developing rat brain and fractionates across a sucrose gradient in a manner similar to synaptophysin, a well-known vesicular and plasma membrane protein. MAP1B is also in axolemma-enriched fractions (AEFs) isolated from myelinated axons of rat brain. MAP1B in AEFs and membrane fractions from cultured dorsal root ganglion neurons (DRGNs) remains membrane-associated following high-salt washes and contains sialic acid. Furthermore, MAP1B in intact DRGNs is readily degraded by extracellular trypsin and is labeled by the cell surface probe sulfosuccinimidobiotin. Immunocytochemical examination of DRGNs shows that MAP1B is concentrated in vesicle-rich varicosities along the length of axons. Myelinated peripheral nerves immunostained for MAP1B show an enrichment at the axonal plasma membrane. These observations demonstrate that some of the MAP1B in developing neurons is an integral plasma membrane glycoprotein.
Topics: Amino Acid Sequence; Animals; Axons; Brain; Cell Membrane; Cells, Cultured; Cerebral Cortex; Fetus; Ganglia, Spinal; Microsomes; Microtubule-Associated Proteins; Molecular Sequence Data; Neurons; Peptide Fragments; Rats; Rats, Sprague-Dawley; Trypsin
PubMed: 10899930
DOI: 10.1046/j.1471-4159.2000.0750553.x -
The Journal of Cell Biology Dec 1999Mice incapable of synthesizing the abundant galactolipids of myelin exhibit disrupted paranodal axo-glial interactions in the central and peripheral nervous systems....
Mice incapable of synthesizing the abundant galactolipids of myelin exhibit disrupted paranodal axo-glial interactions in the central and peripheral nervous systems. Using these mutants, we have analyzed the role that axo-glial interactions play in the establishment of axonal protein distribution in the region of the node of Ranvier. Whereas the clustering of the nodal proteins, sodium channels, ankyrin(G), and neurofascin was only slightly affected, the distribution of potassium channels and paranodin, proteins that are normally concentrated in the regions juxtaposed to the node, was dramatically altered. The potassium channels, which are normally concentrated in the paranode/juxtaparanode, were not restricted to this region but were detected throughout the internode in the galactolipid-defi- cient mice. Paranodin/contactin-associated protein (Caspr), a paranodal protein that is a potential neuronal mediator of axon-myelin binding, was not concentrated in the paranodal regions but was diffusely distributed along the internodal regions. Collectively, these findings suggest that the myelin galactolipids are essential for the proper formation of axo-glial interactions and demonstrate that a disruption in these interactions results in profound abnormalities in the molecular organization of the paranodal axolemma.
Topics: Animals; Ankyrins; Axons; Cell Adhesion Molecules; Cell Communication; Galactolipids; Galactosyltransferases; Ganglioside Galactosyltransferase; Gene Deletion; Glycolipids; Membrane Glycoproteins; Mice; Mice, Knockout; Myelin Sheath; Nerve Growth Factors; Neuroglia; Neuropeptides; Potassium Channels; Ranvier's Nodes; Sciatic Nerve; Sodium Channels; Spinal Cord
PubMed: 10601330
DOI: 10.1083/jcb.147.6.1145 -
Journal of Neurochemistry Nov 1999Schwann cells cloned from rat sciatic nerve survive and display self-induced growth suppression, or undergo spontaneous apoptosis, on long-term serum-free subconfluent...
Schwann cells cloned from rat sciatic nerve survive and display self-induced growth suppression, or undergo spontaneous apoptosis, on long-term serum-free subconfluent culture. Strain SCL4.1/F7 sustained the capacity to growth arrest for up to 40 generations. A soluble activity transmitted between neighbouring cells of this strain suppresses DNA synthesis within three cell cycles. Autocrine Schwann cell growth-inhibitory factor (SGIF) operates during the G1 phase of the cell cycle, overcomes the mitogenic action of Schwann cell/serum-associated (platelet-derived growth factor-BB) and axon-associated (axolemma-enriched fraction) stimuli in serum-free conditions, and suppresses DNA synthesis in sciatic nerve Schwann cell cultures in a stage-specific manner. A 35-kDa protein with N-terminal sequence and approximate molecular mass of the C-propeptide of rat alpha1-procollagen I makes a major contribution to SGIF. Growth suppression in the SCL4.1/F7 strain is mediated by the ras/extracellular signal-regulated kinase pathway, is accompanied by down-regulation of erbB2/erbB3 and of tetraethylammonium-sensitive K+ currents, and is followed by transition of cells within 5-10 days from O4+, p75 nerve growth factor receptor (p75NGF-R)+, glial fibrillary acidic protein (GFAP)+ to O4+, p75NGF-R-, GFAP-, periaxin+ phenotypes. Oct-6/SCIP mRNA is present in both proliferating and growth-arrested SCL4.1/F7 cells. These results demonstrate an autocrine/ paracrine loop for the growth arrest of clonally derived Schwann cells in the absence of axons linked in part to the metabolism of collagen. Schwann cells thus appear to self-regulate growth in a negative as well as a positive direction through characterized molecular mechanisms and signal pathways.
Topics: Animals; Becaplermin; Cell Differentiation; Cell Division; Cells, Cultured; Culture Media, Conditioned; DNA; G1 Phase; Growth Inhibitors; Mitogens; Myelin Sheath; Peptide Fragments; Platelet-Derived Growth Factor; Potassium Channels; Procollagen; Proto-Oncogene Proteins c-sis; Rats; Receptor Protein-Tyrosine Kinases; Receptor, EphA8; Receptor, ErbB-2; Schwann Cells; Sciatic Nerve; Stem Cells; Time Factors
PubMed: 10537039
DOI: No ID Found -
The Journal of Neuroscience : the... Jun 1999Although axonal injury is a common feature of brain trauma, little is known of the immediate morphological responses of individual axons to mechanical injury. Here, we...
Although axonal injury is a common feature of brain trauma, little is known of the immediate morphological responses of individual axons to mechanical injury. Here, we developed an in vitro model system that selectively stretches axons bridging two populations of human neurons derived from the cell line N-Tera2. We found that these axons demonstrated a remarkably high tolerance to dynamic stretch injury, with no primary axotomy at strains <65%. In addition, the axolemma remained impermeable to small molecules after injury unless axotomy had occurred. We also found that injured axons exhibited the behavior of "delayed elasticity" after injury, going from a straight orientation before injury to developing an undulating course as an immediate response to injury, yet gradually recovering their original orientation. Surprisingly, some portions of the axons were found to be up to 60% longer immediately after injury. Subsequent to returning to their original length, injured axons developed swellings of appearance remarkably similar to that found in brain-injured humans. These findings may offer insight into mechanical-loading conditions leading to traumatic axonal injury and into potential mechanisms of axon reassembly after brain trauma.
Topics: Axons; Axotomy; Cells, Cultured; Elasticity; Humans; Neurofilament Proteins; Neurons; Permeability; Reaction Time; Stress, Mechanical
PubMed: 10341230
DOI: 10.1523/JNEUROSCI.19-11-04263.1999 -
Biophysical Journal May 1999To account for the beading of myelinated fibers, and axons of unmyelinated nerve fibers as well of neurites of cultured dorsal root ganglia caused by mild stretching, a...
To account for the beading of myelinated fibers, and axons of unmyelinated nerve fibers as well of neurites of cultured dorsal root ganglia caused by mild stretching, a model is presented. In this model, membrane tension and hydrostatic pressure are the basic factors responsible for axonal constriction, which causes the movement of axonal fluid from the constricted regions into the adjoining axon, there giving rise to the beading expansions. Beading ranges from a mild undulation, with the smallest degree of stretch, to more globular expansions and narrow intervening constrictions as stretch is increased: the degree of constriction is physically limited by the compaction of the cytoskeleton within the axons. The model is a general one, encompassing the possibility that the membrane skeleton, composed mainly of spectrin and actin associated with the inner face of the axolemma, could be involved in bringing about the constrictions and beading.
Topics: Animals; Biophysical Phenomena; Biophysics; Cats; Freeze Substitution; Ganglia, Spinal; In Vitro Techniques; Microscopy, Electron; Models, Neurological; Nerve Fibers; Nerve Fibers, Myelinated; Rats; Sciatic Nerve; Stress, Mechanical
PubMed: 10233101
DOI: 10.1016/S0006-3495(99)77439-4 -
The Journal of Neuroscience : the... Aug 1998Despite the biophysical and clinical importance of differentiating nodal and internodal axolemma, very little is known about the process. We chose to study myelination...
Despite the biophysical and clinical importance of differentiating nodal and internodal axolemma, very little is known about the process. We chose to study myelination and node of Ranvier formation in the hypomyelinating mouse mutant claw paw (clp). The phenotype of clp is delayed myelination in the peripheral nervous system. The specific defect is unknown but is thought to arise from a breakdown in the complex signaling mechanism between axon and Schwann cell. Myelination was assessed in sciatic nerve cross sections from adult and postnatal day 14 (P14) heterozygous and homozygous clp mice. Antibodies to P0, myelin-associated glycoprotein (MAG), and neural cell adhesion molecule were used to assess the stage of myelination. P14 homozygous clp mice showed an atypical staining pattern of immature myelin, which resolved into a relatively normal pattern by adulthood. Sodium channel clustering and node of Ranvier frequency were studied in whole-mount sciatic nerves with sodium channel and MAG antibodies. P14 homozygous clp nerves again showed an atypical, immature pattern with diffuse sodium channel clusters suggesting nodal formation was delayed. In the adult, homozygous clp sciatic nerves displayed dramatically shortened internodal distances. The data from this study support the hypotheses that node of Ranvier formation begins with the onset of myelination and that the number and location of nodes of Ranvier in the sciatic nerve are determined by myelinating Schwann cells.
Topics: Animals; Axons; Biomarkers; Female; Genes, Recessive; Heterozygote; Homozygote; Male; Mice; Mice, Neurologic Mutants; Myelin Sheath; Phenotype; Ranvier's Nodes; Sciatic Nerve; Signal Transduction; Sodium Channels
PubMed: 9671673
DOI: 10.1523/JNEUROSCI.18-15-05859.1998 -
The Journal of Neuroscience : the... Aug 1998The glutamate transporter GLT-1 is expressed in astrocytes of the mature brain and spinal cord. In the present study, we examined its expression in the developing mouse...
The glutamate transporter GLT-1 is expressed in astrocytes of the mature brain and spinal cord. In the present study, we examined its expression in the developing mouse spinal cord. By in situ hybridization, 35S-labeled antisense oligonucleotide probes for GLT-1 mRNA consistently labeled the mantle zone/gray matter from embryonic day 11 through the adult stage. However, immunohistochemistry with a specific antibody visualized distinct regional and cellular localizations during the time between the fetal and postnatal stages. At fetal stages, GLT-1 immunoreactivity predominated in the marginal zone/white matter, observed as tiny puncta in cross-sections and as thin fibers in longitudinal sections. The GLT-1-immunopositive structures were also labeled for neuron-specific enolase, a glycolytic enzyme specific to postmitotic neurons and endocrine cells. By electron microscopy, GLT-1 immunoreactivity was detected in axons forming frequent enlargements and was focally localized on a small portion of the axolemma, particularly that facing adjacent axons. At early postnatal stages, GLT-1 disappeared from axons in white matter tracts and, instead, appeared in astrocytic processes surrounding various neuronal elements in the gray matter. Therefore, before switching to astrocytic expression, GLT-1 is transiently expressed in neurons and localized in differentiating axons. Together with our previous finding on the localization of glutamate transporter GLAST in radial glial fibers, GLT-1 and GLAST are thus localized during development on distinct directional cellular elements along which young neurons elongate their axons or move their cell bodies, respectively.
Topics: ATP-Binding Cassette Transporters; Amino Acid Sequence; Amino Acid Transport System X-AG; Animals; Antibody Formation; Astrocytes; Axons; Biological Transport; Embryonic and Fetal Development; Immunohistochemistry; In Situ Hybridization; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Nerve Tissue Proteins; Neurons; Spinal Cord; Time Factors
PubMed: 9671661
DOI: 10.1523/JNEUROSCI.18-15-05706.1998