myelin-basic-protein and Wallerian-Degeneration

Peripheral myelination is a dynamic process orchestrated by axons and Schwann cells. Although the signaling mechanisms governing myelination are not fully understood, NF-κB activation in Schwann cells has been implicated as a key regulator in vitro. Using a mouse model, we show that nuclear factor κB activation in Schwann cells is not required for myelination in vivo.

Topics: Animals; Animals, Newborn; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Embryo, Mammalian; Gene Expression Regulation, Enzymologic; I-kappa B Kinase; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Myelin P0 Protein; Myelin Sheath; Nerve Growth Factors; NF-kappa B; Proteins; RNA, Untranslated; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Schwann Cells; Sciatic Nerve; Sciatic Neuropathy; Time Factors; Wallerian Degeneration

Neuregulin 1 (NRG1) is an axon-derived factor that is critical for Schwann cell (SC) development and myelinogenesis in a manner dependent on transmembrane tyrosine kinases ErbB2 and ErbB3. Recent studies suggest that NRG1 signaling plays a role in remyelination of regenerated nerves after injury. In this study, we investigated the role of Erbin, a protein that interacts with ErbB2 in remyelination of injured nerves. We show that Erbin expression increased dramatically in injured nerves. Myelinated axons were fewer, and g-ratios of those that were myelinated were increased in erbin(-/-) mice, which were impaired in functional recovery from nerve injury. These results indicate a necessary role of Erbin in remyelination of regenerating axons. Erbin ablation had little effect on numbers of BrdU-labeled and TUNEL-labeled SCs, suggesting mechanisms independent of altered proliferation or apoptosis. We demonstrated that Erbin mutant mice were impaired in raising or maintaining the levels of ErbB2 and in producing NRG1 in axons. Together, these observations demonstrate that Erbin is required for remyelination of regenerated axons after injury, probably by regulating ErbB2 and NRG1 levels, identifying a novel player in regulating remyelination.

Topics: Animals; Axons; Bromodeoxyuridine; Carrier Proteins; Cell Death; Disease Models, Animal; Female; Gene Expression Regulation; In Situ Nick-End Labeling; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microscopy, Electron, Transmission; Myelin Basic Protein; Myelin Sheath; Nerve Regeneration; Neuregulin-1; Receptor, ErbB-2; Receptor, ErbB-3; Recovery of Function; RNA, Messenger; Sciatic Neuropathy; Time Factors; Wallerian Degeneration

ABSTRACT Diffusion tensor imaging (DTI) and quantitative T(2) magnetic resonance imaging (MRI) were used to characterize ex vivo the white matter damage at 3 and 8 weeks following dorsal column transection (DC Tx) injury at the cervical level C5 of rat spinal cords. Luxol Fast Blue (LFB) and myelin basic protein (MBP) staining was used to assess myelin damage, and neurofilament-H in combination with neuron specific beta-III-tubulin (NF/Tub) staining was used to assess axonal damage. Average values of myelin water fraction (MWF), fractional anisotropy (FA), longitudinal diffusivity (D(long)), transverse diffusivity (D(trans)), and average diffusivity (D(ave)) were calculated in the fasciculus gracilis, fasciculus cuneatus, and the dorsal corticospinal tract (CST) 5 mm cranial, as well as 5 and 10 mm caudal to injury and correlated with histology. These tracts were selected as these contain bundles of parallel ascending and descending axons in very circumscribed areas with little intermingling of other axonal populations. Axonal and myelin degeneration occur cranial to injury in the funiculus gracilis and caudal to injury in the CST. Both MWF and D(trans) showed significant correlation with LFB staining at 3 weeks (0.64 and -0.49, respectively) and 8 weeks post-injury (0.88 and -0.71, respectively). Both D(long) and FA correlated significantly with NF/Tub staining at 3 weeks post-injury (0.78 and 0.64, respectively), while only D(long) displayed significant correlation 8 weeks post-injury (0.58 and 0.33, respectively). This study demonstrates that quantitative MRI can accurately characterize white matter damage in DC Tx model of injury in rat spinal cord.

Topics: Animals; Anisotropy; Biomarkers; Body Water; Diffusion; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Disease Progression; Immunohistochemistry; Male; Myelin Basic Protein; Nerve Fibers, Myelinated; Neural Pathways; Neurofilament Proteins; Predictive Value of Tests; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Staining and Labeling; Time Factors; Trauma Severity Indices; Tubulin; Wallerian Degeneration

Hypoxia-ischemia (H/I) in the premature infant leads to white matter injury termed periventricular leukomalacia (PVL), the leading cause of subsequent neurological deficits. Glutamatergic excitotoxicity in white matter oligodendrocytes (OLs) mediated by cell surface glutamate receptors (GluRs) of the AMPA subtype has been demonstrated as one factor in this injury. Recently, it has been shown that rodent OLs also express functional NMDA GluRs (NMDARs), and overactivation of these receptors can mediate excitotoxic OL injury. Here we show that preterm human developing OLs express NMDARs during the PVL period of susceptibility, presenting a potential therapeutic target. The expression pattern mirrors that seen in the immature rat. Furthermore, the uncompetitive NMDAR antagonist memantine attenuates NMDA-evoked currents in developing OLs in situ in cerebral white matter of immature rats. Using an H/I rat model of white matter injury, we show in vivo that post-H/I treatment with memantine attenuates acute loss of the developing OL cell surface marker O1 and the mature OL marker MBP (myelin basic protein), and also prevents the long-term reduction in cerebral mantle thickness seen at postnatal day 21 in this model. These protective doses of memantine do not affect normal myelination or cortical growth. Together, these data suggest that NMDAR blockade with memantine may provide an effective pharmacological prevention of PVL in the premature infant.

Topics: Animals; Animals, Newborn; Antigens, Differentiation; Biomarkers; Brain; Disease Models, Animal; Excitatory Amino Acid Antagonists; Humans; Hypoxia-Ischemia, Brain; Infant, Newborn; Leukomalacia, Periventricular; Male; Memantine; Myelin Basic Protein; Nerve Fibers, Myelinated; Oligodendroglia; Rats; Rats, Long-Evans; Receptors, N-Methyl-D-Aspartate; Wallerian Degeneration

Transgenic and disease model mice have been used to investigate the molecular mechanisms of demyelinating diseases. However, less attention has been given to elucidating changes in nerve conduction in these mice. We established an experimental system to measure the response latency of cortical neurons and examined changes in nerve conduction in cuprizone-induced demyelinating mice and in myelin basic protein-deficient shiverer mice. Stimulating and recording electrodes were placed in the right and left sensori-motor cortices, respectively. Electrical stimulation of the right cortex evoked antidromic responses in left cortical neurons with a latency of 9.38 +/- 0.31 ms (n = 107; mean +/- SEM). While response latency was longer in mice at 7 days and 4 weeks of cuprizone treatment (12.35 +/- 0.35 ms, n = 102; 11.72 +/- 0.29 ms, n = 103, respectively), response latency at 7 days and 4 weeks after removal of cuprizone was partially restored (10.72 +/- 0.45 ms, n = 106; 10.27 +/- 0.34 ms, n = 107, respectively). Likewise, electron microscopy showed cuprizone-induced demyelination in the corpus callosum and nearly complete remyelination after cuprizone removal. We also examined whether the myelin abnormalities in shiverer mice affected their response latencies. But there were no significant differences in response latencies in shiverer (9.83 +/- 0.24 ms, n = 103) and wild-type (9.33 +/- 0.22 ms, n = 112) mice. The results of these electrophysiological assessments imply that different demyelinating mechanisms, differentially affecting axon conduction, are present in the cuprizone-treated and shiverer mice, and may provide new insights to understanding the pathophysiology of demyelination in animal models in the CNS.

Topics: Animals; Axons; Central Nervous System; Chelating Agents; Cuprizone; Demyelinating Diseases; Disease Models, Animal; Electric Stimulation; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Neurologic Mutants; Motor Cortex; Myelin Basic Protein; Nerve Fibers, Myelinated; Neural Conduction; Neural Pathways; Reaction Time; Wallerian Degeneration

Wallerian degeneration plays a significant role in many central nervous system (CNS) diseases. Tracking the progression of Wallerian degeneration may provide better understanding of the evolution of many CNS diseases. In this study, a 28-day longitudinal in vivo DTI of optic nerve (ON) and optic tract (OT) was conducted to evaluate the temporal and spatial evolution of Wallerian degeneration resulting from the transient retinal ischemia. At 3-28 days after ischemia, ipsilateral ON and contralateral OT showed significant reduction in axial diffusivity (32-40% and 21-29% respectively) suggestive of axonal damage. Both ON and OT showed significant increase in radial diffusivity, 200-290% and 58-65% respectively, at 9-28 days suggestive of myelin damage. Immunohistochemistry of phosphorylated neurofilament (pNF) and myelin basic protein (MBP) was performed to assess axonal and myelin integrities validating the DTI findings. Both DTI and immunohistochemistry detected that transient retinal ischemia caused more severe damage to ON than to OT. The current results suggest that axial and radial diffusivities are capable of reflecting the severity of axonal and myelin damage in mice as assessed using immunohistochemistry.

Topics: Algorithms; Animals; Axons; Cell Count; Cell Death; Diffusion Magnetic Resonance Imaging; Immunohistochemistry; Male; Mice; Myelin Basic Protein; Myelin Sheath; Neurofilament Proteins; Optic Nerve; Phosphorylation; Retinal Ganglion Cells; Retinal Vessels; Visual Pathways; Wallerian Degeneration

The pathogenic mechanisms that contribute to multiple sclerosis (MS) include leukocyte chemotaxis into the central nervous system (CNS) and the production of inflammatory mediators, resulting in oligodendrocyte damage, demyelination, and neuronal injury. Thus, factors that regulate leukocyte entry may contribute to early events in MS, as well as to later stages of lesion pathogenesis. CXCL12 (SDF-1alpha), a chemokine essential in CNS development and a chemoattractant for resting and activated T cells, as well as monocytes, is constitutively expressed at low levels in the CNS and has been implicated in T cell and monocyte baseline trafficking. To determine whether CXCL12 is increased in MS, immunohistochemical analyses of lesions of chronic active and chronic silent MS were performed. CXCL12 protein was detected on endothelial cells (EC) in blood vessels within normal human brain sections and on a small number of astrocytes within the brain parenchyma. In active MS lesions, CXCL12 levels were high on astrocytes throughout lesion areas and on some monocytes/macrophages within vessels and perivascular cuffs, with lesser staining on EC. In silent MS lesions, CXCL12 staining was less than that observed in active MS lesions, and also was detected on EC and astrocytes, particularly hypertrophic astrocytes near the lesion edge. Experiments in vitro demonstrated that IL-1beta and myelin basic protein (MBP) induced CXCL12 in astrocytes by signaling pathways involving ERK and PI3-K. Human umbilical vein EC did not produce CXCL12 after treatment with MBP or IL-1beta. However, these EC cultures expressed CXCR4, the receptor for CXCL12, suggesting that this chemokine may activate EC to produce other mediators involved in MS. In agreement, EC treatment with CXCL12 was found to upregulate CCL2 (MCP-1) and CXCL8 (IL-8) by PI3-K and p38-dependent mechanisms. Our findings suggest that increased CXCL12 may initiate and augment the inflammatory response during MS.

Topics: Adolescent; Adult; Aged, 80 and over; Astrocytes; Axons; Cells, Cultured; Central Nervous System; Chemokine CCL2; Chemokine CXCL12; Chemokines, CXC; Chemotaxis, Leukocyte; Endothelial Cells; Female; Humans; Interleukin-1; Interleukin-8; Macrophages; Male; MAP Kinase Signaling System; Middle Aged; Multiple Sclerosis; Myelin Basic Protein; Myelin Sheath; Receptors, CXCR4; Wallerian Degeneration

In this work, we have immunohistochemically analyzed the effects of single injections of apotransferrin (aTf) on the expression of myelin (myelin basic proteins [MBPs]) and axonal (protein gene product 9.5 [PGP 9.5] and beta(III)-tubulin [beta(III)-tub]) proteins in colchicine-injected and crushed sciatic nerves of adult rats. A protein redistribution was seen in the distal stump of injured nerves, with the appearance of MBP- and PGP 9.5-immunoreactive (IR) clusters which occurred earlier in crushed nerves (3 days post-injury [PI]) as compared to colchicine-injected nerves (7 days PI). beta(III)-tub-IR clusters appeared at 1 day PI preceding the PGP 9.5- and MBP-IR clusters in colchicine-injected nerves. With image analysis, the peak of clustering formation was found at 14 days PI for MBP and at 3 days PI for beta(III)-tub in colchicine-injected nerves. At 28 days of survival, the protein distribution patterns were almost normal. The intraneural application of aTf, at different concentrations (0.0005 mg/ml, 0.005 mg/ml, 0.05 mg/ml, 0.5 mg/ml), prevented nerve degeneration produced by colchicine, with the appearance of only a small number of MBP- and beta(III)-tub-IR clusters. However, aTf was not able to prevent clustering formation when the nerve was crushed, a kind of injury that also involves necrosis and blood flow alterations. The results suggest that aTf could prevent the colchicine effects by stabilizing the cytoskeleton proteins of the nerve fibers, avoiding the disruption of the axonal transport and thus the myelin degeneration. Transferrin is proposed as a complementary therapeutic avenue for treatment of cytotoxic nerve injuries.

Topics: Animals; Apoproteins; Axonal Transport; Axons; Colchicine; Cytoskeleton; Disease Models, Animal; Dose-Response Relationship, Drug; Immunohistochemistry; Microtubules; Myelin Basic Protein; Myelin Sheath; Nerve Tissue Proteins; Neuroprotective Agents; Neurotoxins; Rats; Rats, Wistar; Sciatic Neuropathy; Transferrin; Treatment Outcome; Tubulin; Ubiquitin Thiolesterase; Wallerian Degeneration

Dithiocarbamates (DTCs), such as disulfiram, have been used in aversion therapy for alcoholism even though an inherent toxicity is induced, which is related mainly to peripheral neuropathy and is associated with behavioural and neurological complications. At anatomical and histopathological levels, DTCs affect structural elements in nervous tissue, such as axonal degeneration and alterations in the cytoskeletal proteins of astrocytes. Therefore, given the axonal effects of DTCs and to gain further insight into axonal growth and axonal pathfinding in the central nervous system (CNS), here we established an in vivo experimental model of mouse development. Daily intraperitoneal injections of N,N-diethyldithiocarbamate (DEDTC), the first metabolite of disulfiram, were given from postnatal day 2 (P2) until P15. From P16 until P30, animals were not treated. Treatment induced considerable physiological alterations, such as growth delay, throughout postnatal development. Moreover, by immunohistochemistry techniques, we observed important alterations in the cytoskeletal glial protein at early stages of postnatal development. At later stages (P15), the immunoreactivity pattern detected by an antibody against axonal neurofilaments (anti-NF-H) showed alteration in the axonal distribution pattern followed by drastic axonal loss at P22, data that were corroborated using an anti-MBP (myelin basic protein) antibody. Using an antibody against the beta amyloid precursor protein (APP), we detected axonal injury. Furthermore, given that we observed axonal re-growth in adulthood in the in vivo model presented, we propose that this model would be a good system in which to identify new strategies for inducing regenerative growth in neural diseases in which axonal regeneration is blocked.

Topics: Aging; Amyloid beta-Protein Precursor; Animals; Animals, Newborn; Antidotes; Axons; Cell Differentiation; Central Nervous System; Cytoskeletal Proteins; Disease Models, Animal; Ditiocarb; Growth Cones; Immunohistochemistry; Mice; Myelin Basic Protein; Nerve Degeneration; Nerve Regeneration; Neurofilament Proteins; Neurotoxins; Wallerian Degeneration

How do myelinated axons signal to the nuclei of cells that enwrap them? The cell bodies of oligodendrocytes and Schwann cells are segregated from axons by multiple layers of bimolecular lipid leaflet and myelin proteins. Conventional signal transduction strategies would seem inadequate to the challenge without special adaptations. Wallerian degeneration provides a model to study axon-to-Schwann cell signaling in the context of nerve injury. We show a hitherto undetected rapid, but transient, activation of the receptor tyrosine kinase erbB2 in myelinating Schwann cells after sciatic nerve axotomy. Deconvolving microscopy using phosphorylation state-specific antibodies shows that erbB2 activation emanates from within the microvilli of Schwann cells, in direct contact with the axons they enwrap. To define the functional role of this transient activation, we used a small molecule antagonist of erbB2 activation (PKI166). The response of myelinating Schwann cells to axotomy is inhibited by PKI166 in vivo. Using neuron/Schwann cell cocultures prepared in compartmentalized cell culture chambers, we show that even transient activation of erbB2 is sufficient to initiate Schwann cell demyelination and that the initiating functions of erbB2 are localized to Schwann cells.

Topics: Analysis of Variance; Animals; Axotomy; Blotting, Western; Bromodeoxyuridine; Cell Proliferation; Cells, Cultured; Coculture Techniques; Demyelinating Diseases; Disease Models, Animal; Embryo, Mammalian; Female; Fluorescent Antibody Technique; Ganglia, Spinal; Gene Expression; Glycoproteins; Immunoprecipitation; Mitogen-Activated Protein Kinase Kinases; Myelin Basic Protein; Myelin Sheath; Neuregulins; Neuroglia; Neurons; Platelet-Derived Growth Factor; Pyrimidines; Pyrroles; Rats; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Receptor, ErbB-2; Schwann Cells; Sciatic Neuropathy; Signal Transduction; Sodium Channels; Time Factors; Wallerian Degeneration

The inflammatory cytokine tumour necrosis factor (TNF) can both induce oligodendrocyte and myelin pathology and promote proliferation of oligodendrocyte progenitor cells and remyelination. We have compared the response of the oligodendrocyte lineage to anterograde axonal (Wallerian) and terminal degeneration and lesion-induced axonal sprouting in the hippocampal dentate gyrus in TNF-transgenic mice with the response in genetically normal mice. Transectioning of the entorhino-dentate perforant path axonal projection increased hippocampal TNF mRNA expression in both types of mice, but to significantly larger levels in the TNF-transgenics. At 5 days after axonal transection, numbers of oligodendrocytes and myelin basic protein (MBP) mRNA expression in the denervated dentate gyrus in TNF-transgenic mice had increased to the same extent as in nontransgenic littermates. At this time, transgenics showed a tendency towards a greater increase in the number of juxtaposed, potentially proliferating oligodendrocytes. Noteworthy, at day 5 we also observed upregulation of MBP mRNA expression in adjacent hippocampal subregions with lesion-induced axonal sprouting, which were devoid of axonal degeneration, raising the possibility that sprouting axons provide trophic stimuli to the oligodendrocyte lineage. Twenty-eight days after lesioning, oligodendrocyte numbers and MBP mRNA expression were reduced to near normal levels. However, oligodendrocyte densities in the TNF-transgenic mice were significantly lower than in nontransgenics. We conclude that the early response of the oligodendrocyte lineage to axonal lesioning and lesion-induced axonal sprouting appears unaffected by the supranormal TNF levels in the TNF-transgenic mice. TNF may, however, have long-term inhibitory effects on the oligodendrocyte response to axonal lesioning.

Topics: Animals; Axons; Axotomy; Cell Division; Hippocampus; Mice; Mice, Transgenic; Myelin Basic Protein; Nerve Fibers, Myelinated; Nerve Growth Factors; Nerve Regeneration; Neuronal Plasticity; Oligodendroglia; Perforant Pathway; Reaction Time; RNA, Messenger; Tumor Necrosis Factor-alpha; Up-Regulation; Wallerian Degeneration

The PLP1/Plp gene encodes proteolipid protein (PLP) and DM20, the major central nervous system myelin proteins. Mutations in the PLP1/ Plp gene cause dysmyelinating disorders in man and animals. The rumpshaker mutation was first identified in mice and later linked to a family diagnosed with neurological deficits akin to spastic paraplegia. The dysmyelination in the young rumpshaker mouse is well characterised. Here we report evidence for an age-related increase in myelin due mainly to the myelination of small axons, many large axons remain dysmyelinated. Levels of PLP/DM20 and myelin basic protein are considerably greater in myelin fractions from older compared with younger mutants. Myelin in sheaths of larger axons remains poorly compacted and may account for levels of 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein being elevated over wild type in older mutant mice. A late-onset distal degeneration of the axons of the longest spinal tract, the fasciculus gracilis, is also noted. This is the first report of Wallerian-type degeneration in mice with spontaneous mutations of the Plp gene.

Topics: Age Factors; Animals; Animals, Newborn; Axons; Blotting, Western; Brain; Immunohistochemistry; Mice; Mice, Mutant Strains; Microscopy, Electron; Mutation; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Spinal Cord; Wallerian Degeneration

Multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS, are inflammatory demyelinating diseases of the central nervous system. The inflammatory attacks lead to glial dysfunction and death, axonal damage, and neurological deficits. Numerous studies in rat suggest that extracellular calcium influx, via voltage-gated calcium channels (VGCC), contributes to white matter damage in acute spinal cord injury and stroke. Our immunohistochemical finding that mouse spinal cord axons display subunits of L-type VGCC also supports this hypothesis. Furthermore, we hypothesized that VGCC also play a role in EAE, and possibly, MS. In our study, administration of the calcium channel blockers (CCB) bepridil and nitrendipine significantly ameliorated EAE in mice, compared with vehicle-treated controls. Spinal cord samples showed reduced inflammation and axonal pathology in bepridil-treated animals. Our data support the hypothesis that calcium influx via VGCC plays a significant role in the development of neurological disability and white matter damage in EAE and MS.

Topics: Animals; Axons; Bepridil; Calcium Channel Blockers; Calcium Channels, L-Type; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Female; Immunohistochemistry; Mice; Multiple Sclerosis; Myelin Basic Protein; Neutrophil Infiltration; Nitrendipine; Spinal Cord; Time Factors; Wallerian Degeneration

Axotomy of nerve fibers leads to the subsequent degeneration of their distal part, a process termed Wallerian degeneration (WD). While WD in the peripheral nervous system is usually followed by regeneration of the lesioned axons, central nervous system (CNS) neurons are generally unable to regrow. In this study, we investigated the process of WD in the dorsal columns of the rat spinal cord rostral to a mid-thoracic lesion. We confirm earlier studies describing a very delayed microglial and an early and sustained astroglial reaction finally leading to scar formation. Interestingly, we found a differential time course in the loss of myelin proteins depending on their location. Proteins situated on the periaxonal myelin membrane such as myelin associated glycoprotein disappeared early, within a few days after lesion, concomitantly with cytoskeletal axonal proteins, whereas compact myelin and outer myelin membrane proteins such as MBP and Nogo-A remained for long intervals in the degenerating tracts. Two distinct mechanisms are probably responsible for this difference: processes of protein destruction emanating from and initially probably located in the axon act on a time scale of 1-3 days. In contrast, the bulk of myelin destruction is due to phagocytosis known to be slow, prolonged, and inefficient in the CNS. These results may also have implications for future intervention strategies aiming at enhancing CNS regeneration.

Topics: Animals; Astrocytes; Cell Survival; Female; Myelin Basic Protein; Myelin Proteins; Myelin-Associated Glycoprotein; Nerve Regeneration; Neurofilament Proteins; Nogo Proteins; Rats; Rats, Inbred Lew; Spinal Cord; Wallerian Degeneration

Both axon and myelin degeneration have significant impact on the long-term disability of patients with white matter disorder. However, the clinical manifestations of the neurological dysfunction caused by white matter disorders are not sufficient to determine the origin of neurological deficits. A noninvasive biological marker capable of detecting and differentiating axon and myelin degeneration would be a significant addition to currently available tools. Directional diffusivities derived from diffusion tensor imaging (DTI) have been previously proposed by this group as potential biological markers to detect and differentiate axon and myelin degeneration. To further test the hypothesis that axial (lambdaparallel) and radial (lambdaperpendicular) diffusivities reflect axon and myelin pathologies, respectively, the optic nerve was examined serially using DTI in a mouse model of retinal ischemia. A significant decrease of lambdaparallel, the putative DTI axonal marker, was observed 3 days after ischemia without concurrently detectable changes in lambdaperpendicular, the putative myelin marker. This result is consistent with histological findings of significant axonal degeneration with no detectable demyelination at 3 days after ischemia. The elevation of lambdaperpendicular observed 5 days after ischemia is consistent with histological findings of myelin degeneration at this time. These results support the hypothesis that lambdaparallel and lambdaperpendicular hold promise as specific markers of axonal and myelin injury, respectively, and, further, that the coexistence of axonal and myelin degeneration does not confound this utility.

Topics: Animals; Axons; Diffusion Magnetic Resonance Imaging; Immunohistochemistry; Ischemia; Mice; Models, Neurological; Myelin Basic Protein; Myelin Sheath; Nerve Degeneration; Neurofilament Proteins; Optic Nerve; Retinal Degeneration; Retinal Vessels; Wallerian Degeneration

Cerebral white matter (WM) lesions are observed frequently in human ischemic cerebrovascular disease and have been thought to contribute to cognitive impairment. This type of lesion can be experimentally induced in rat brains under chronic cerebral hypoperfusion by the permanent occlusion of both common carotid arteries. However, it remains uncertain whether chronic ischemia can damage both the gray and white matter, and whether it can induce demyelination with or without axonal damage. Therefore, we examined axonal damage using immunohistochemistry for the amyloid beta/A4 precursor protein (APP), chromogranin A (CgA) and demyelination using immunohistochemistry for the encephalitogenic peptide (EP) in this model. Severe WM lesions such as vacuolation and the loss of nerve fibers appeared in the optic nerve and optic tract after 3 days of ligation, and less intense changes were observed in the corpus callosum, internal capsule, and fiber bundles of the caudoputamen after 7 days with Klüver-Barrera and Bielschowsky staining. These WM lesions persisted even after 30 days. The APP, CgA, and EP-immunopositive fibers increased in number from 1 to 30 days after the ligation in the following WM regions: the optic nerve, optic tract, corpus callosum, internal capsule, and fiber bundles of the caudoputamen. In contrast, only a few APP, CgA, or EP-immunopositive fibers were detected in the gray matter regions, including the cerebral cortex and hippocampus. These results indicate that the WM is more susceptible to chronic cerebral hypoperfusion than the gray matter, with an involvement of both axonal and myelin components. Furthermore, immunohistochemistry for APP, CgA, and EP is far superior to routine histological staining in sensitivity and may become a useful tool to investigate WM lesions caused by various pathoetiologies.

Topics: Amyloid beta-Protein Precursor; Animals; Axons; Brain Ischemia; Cerebral Infarction; Chromogranin A; Chromogranins; Chronic Disease; Demyelinating Diseases; Immunohistochemistry; Male; Myelin Basic Protein; Nerve Fibers, Myelinated; Peptide Fragments; Prosencephalon; Rats; Rats, Wistar; Wallerian Degeneration

Macrophages play a central role in the pathogenesis of peripheral neuropathy but the role of resident endoneurial macrophages is undefined because no discriminating markers exist to distinguish them from infiltrating hematogenous macrophages. We identified and characterized resident endoneurial macrophages during Wallerian degeneration in radiation bone marrow chimeric rats created by transplanting wild-type Lewis rat bone marrow into irradiated TK-tsa transgenic Lewis rats. In such animals, resident cells carry the transgene, whereas hematogenous cells do not. As early as 2 days after sciatic nerve crush and before the influx of hematogenous macrophages, resident transgene-positive endoneurial macrophages underwent morphological and immunophenotypic signs of activation. At the same time, resident macrophages phagocytosing myelin were found, and proliferation was detected by bromodeoxyuridine incorporation. Continuous bromodeoxyuridine feeding revealed that resident endoneurial macrophages sequentially retracted their processes, proliferated, and expressed the ED1 antigen, rendering them morphologically indistinguishable from hematogenous macrophages. Resident endoneurial macrophages thus play an early and active role in the cellular events after nerve lesion before hematogenous macrophages enter the nerve. They may thus be critically involved in the pathogenesis of peripheral neuropathy particularly at early stages of the disease and may act as sensors of pathology much like their central nervous system counterparts, the microglial cells.

Topics: Animals; Animals, Genetically Modified; Antigens; Cell Division; Immunohistochemistry; In Situ Hybridization; Macrophages; Myelin Basic Protein; Myelin Sheath; Phagocytosis; Radiation Chimera; Rats; Rats, Inbred Lew; Sciatic Nerve; Sensitivity and Specificity; Transgenes; Wallerian Degeneration

We have examined the role of complement component 5 (C5) in peripheral nerve fiber degeneration and regeneration, as well as in glial and neuronal cell responses in the central nervous system (CNS). Adult congenic mice lacking C5 (C5(-)) and the corresponding normal strain (C5(+)) were used. Macrophage recruitment as well as axonal and myelin sheath elimination were delayed from 1 to 21 days postinjury in C5(-) mice compared to the C5(+) group after sciatic nerve crush. Despite this, recovery of motor function was not delayed. In the CNS, microglial cells and astrocytes responded in the same way from 3 to 21 days after sciatic nerve injury in C5(-) and C5(+) mice, and the extent of neuron death following hypoglossal nerve avulsion was the same in both groups. These findings suggest that C5 and/or its derivatives play an important role in initiating the recruitment of macrophages to the injured nerve and, probably indirectly, in early remyelination of regenerating axons, but does not influence the longterm functional restoration or axotomy-induced nerve cell death. C5-derived molecules do not appear to participate in central glial cell responses to peripheral nerve injury. These findings elucidate new aspects on the functional role of the complement system in the peripheral nervous system following peripheral nerve injury.

Topics: Animals; Complement C5; Facial Nerve; Glial Fibrillary Acidic Protein; Hypoglossal Nerve; Mice; Mice, Inbred Strains; Mice, Knockout; Mice, Mutant Strains; Myelin Basic Protein; Nerve Crush; Reference Values; Sciatic Nerve; Wallerian Degeneration

We have investigated the temporal and spatial profiles of apoptotic cells in an experimental transection spinal cord injury by the terminal deoxynucleotidyl transferase-mediated biotin-16-2'-deoxyuridine-5'-triphosphate nick-end labeling (TUNEL) method. Twenty-four hours postinjury, a numerous TUNEL-positive cells appeared both rostrally and caudally to the transection site. Those positive cells, however, gradually diminished in number by several days postinjury. In contrast, other TUNEL-positive cells were found scattered within the white matter remote from the lesion by the third day postinjury. These cells were typically embedded in or among vacuolated fibers, where they were identified in close proximity to the vacuolated space enclosed by myelin basic protein (MBP)-positive structures confirmed by TUNEL-MBP double staining. Because of their linear arrangement, these TUNEL-positive cells were considered interfascicular oligodendrocytes, a fact that was confirmed by the finding that some TUNEL-positive cells were also stained with CCI, a cell marker for oligodendrocyte. Electron microscopic studies revealed that the cells expressing apoptotic morphology were invariably encased in a space formed by myelin splitting. Although the biological significance of apoptotic interfascicular oligodendrocytes in the process of wallerian degeneration is yet to be determined, the finding of such profiles localized within degenerating myelin structures suggests that; oligodendrocytes may be "trapped" within rapidly swollen and disintegrating myelin lamellae, which isolates and perhaps predisposes them to death.

Topics: Animals; Apoptosis; Glial Fibrillary Acidic Protein; Immunohistochemistry; In Situ Nick-End Labeling; Male; Myelin Basic Protein; Nerve Fibers, Myelinated; Neurofilament Proteins; Oligodendroglia; Rats; Rats, Wistar; Spinal Cord; Spinal Cord Injuries; Time Factors; Wallerian Degeneration

The glia response to Wallerian degeneration was studied in optic nerves 21 days after unilateral enucleation (PED21) of immature rats, 21 days old (P21), using immunohistochemical labelling. Nerves from normal P21 and P42 nerves were also studied for comparison. At PED21, there was a virtual loss of axons apart from a few solitary fibres of unknown origin. The nerve comprised a homogeneous glial scar tissue formed by dense astrocyte processes, oriented parallel to the long axis of the nerve along the tracks of degenerated axons. Astrocytes were almost perfectly co-labelled by antibodies to glial fibrillary acid protein and vimentin in both normal and transected nerves. However, there was a small population of VIM+GFAP- cells in normal P21 and P42 nerves, and we discuss the possibility that they correspond to O-2A progenitor cells described in vitro. Significantly, double immunofluorescence labelling in transected nerves revealed a distinct population of hypertrophic astrocytes which were GFAP+VIM-. These cells represented a novel morphological and antigenic subtype of reactive astrocyte. It was also noted that the number of oligodendrocytes in transected nerves did not appear to be less than in normal nerves, on the basis of double immunofluorescence staining for carbonic anhydrase II, myelin oligodendrocyte glycoprotein, myelin basic protein, glial fibrillary acid protein and ED-1 (for macrophages), although it was not excluded that a small proportion may have been microglia. A further prominent feature of transected nerves was that they contained a substantial amount of myelin debris, notwithstanding that OX-42 and ED1 immunostaining showed that there were abundant microglia and macrophages, sufficient for the rapid and almost complete removal of axonal debris. In conclusion, glial cells in the immature P21 rat optic nerve reacted to Wallerian degeneration in a way equivalent to the adult CNS, i.e. astrocytes underwent pronounced reactive changes and formed a dense glial scar, oligodendrocytes persisted and were not dependent on axons for their continued survival, and there was ineffective phagocytosis of myelin possibly due to incomplete activation of microglia/macrophages.

Topics: Age Factors; Animals; Animals, Newborn; Astrocytes; Axons; Eye Enucleation; Fluorescent Dyes; Glial Fibrillary Acidic Protein; Immunohistochemistry; In Vitro Techniques; Microglia; Myelin Basic Protein; Myelin Proteins; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; Neuroglia; Oligodendroglia; Optic Nerve; Rats; Rats, Wistar; Vimentin; Wallerian Degeneration

To study whether nervous tissue trauma provokes myelin antigen autoreactive T and B cell responses in humans we examined consecutive blood samples from 7 patients with polyneuropathy undergoing diagnostic sural nerve biopsy and 8 control patients undergoing other types of minor surgery. The antigen-specific T cells were assessed by enumerating cells secreting interferon-gamma (IFN-gamma) in response to the myelin components P0, P2, myelin basic protein (MBP) and myelin associated glycoprotein (MAG), and to 4 selected MBP peptides. B cell mediated immunity was assessed by counting numbers of cells secreting antibodies directed against the myelin proteins. On day 7 after biopsy, there were 3-10-fold increased numbers of T and B cells reactive with P0, P2, MBP and MAG in blood of polyneuropathy patients compared to controls, while levels of cells recognizing purified protein derivate or responding to phytohemagglutinin (PHA) did not differ significantly. Comparison of prebiopsy levels on day 0 with post-biopsy levels on day 7 in the polyneuropathy patients revealed a significant increase in T cells recognizing P0, P2 and MAG, and in B cells secreting IgG antibodies against P0 and P2. On day 14 after nerve biopsy these differences were no longer seen. We suggest that in patients with polyneuropathy, sural nerve biopsy with the ensuing wallerian degeneration and myelin breakdown causes transiently increased levels of circulating myelin autoreactive T and B cells. It remains to be determined if this has a physiological role in nerve trauma responses and/or affects the clinicopathological course of the peripheral neuropathy.

Topics: Adolescent; Aged; Autoantibodies; Autoimmunity; B-Lymphocytes; Biopsy; Female; Humans; Immunoglobulin G; Interferon-gamma; Male; Middle Aged; Myelin Basic Protein; Myelin P0 Protein; Myelin P2 Protein; Myelin Proteins; Myelin Sheath; Myelin-Associated Glycoprotein; Peptide Fragments; Peripheral Nervous System Diseases; Sural Nerve; T-Lymphocytes; Time Factors; Wallerian Degeneration

It has previously been shown in the adult rat optic nerve that cells with many features of oligodendrocytes are capable of surviving for extended periods of time in the absence of axons. This is in contrast to the situation in the developing nervous system, where removal of axons leads to the failure of differentiation and to the death of oligodendrocytes. In the adult, these surviving oligodendrocytes were not typical in their appearance, and could only be identified with certainty using cell specific markers. In the present experiments, the functional capacity of these long-term quiescent cells to regenerate and myelinate was tested using the Shiverer mouse, a mutant lacking the gene for myelin basic protein (MBP), as a host animal. Fragments of optic nerve from adult rats which had been enucleated up to 2 years previously, were implanted into neonatal Shiverer mice. Four weeks later, the brains were removed and the formation of myelin investigated with antibodies to MBP, to ensure that this was of donor origin. Axons were demonstrated to have grown into the implants, and may have provided the stimulus for the production of MBP by the oligodendrocytes, which were stained positively within the implant. Myelin was demonstrated both within and adjacent to the implant. This study indicates that in the adult central nervous system, cells can survive for extended periods of time in the absence of axons, albeit in an inactive state, and are then capable of functional regeneration when placed in contact with unmyelinated axons. The origin of these cells, either from surviving oligodendrocytes which had previously myelinated the axons, or from progenitors lying within the adult nerve is unclear. The implications of these results are of importance in the further investigation of the potential for central nervous system regeneration.

Topics: Animals; Axons; Brain; Brain Tissue Transplantation; Demyelinating Diseases; Immunohistochemistry; Male; Mice; Mice, Neurologic Mutants; Microscopy, Immunoelectron; Myelin Basic Protein; Myelin Sheath; Oligodendroglia; Optic Nerve; Rats; Transplantation, Heterologous; Wallerian Degeneration

Age-related differences in proliferative responses of Schwann cells during Wallerian degeneration were investigated in the mouse sciatic nerves after nerve-transection at 3, 10 and 60 days of age, corresponding to the periods of early myelination, active myelination and post-myelination. As assessed by thymidine incorporation for the first 24 h in culture, Schwann cells from adult nerve proliferated rapidly within day 1 post-transection and reached a peak at day 3. In the nerves from neonatal or suckling mice, however, division rate of Schwann cells declined after transection, and was even less in the transected nerves than in the contralateral uninjured nerves. The reduction in thymidine uptake by Schwann cells was more pronounced in nerves sectioned at postnatal day 3 than those sectioned at day 10. By contrast, fibroblasts divided rapidly following transection regardless of age. These data suggest that mitogens from myelin components are important for proliferation of Schwann cells and that in the degenerating nerves of young mice, mitotic capacity of Schwann cells declined due to not only a loss of axonal mitogens but also the paucity of mitogens from myelin components. Proliferation of fibroblasts is likely to be stimulated by more general growth-promoting polypeptides common to any other tissues during wound repair.

Topics: Aging; Animals; Animals, Newborn; Autoradiography; Cell Division; Cells, Cultured; DNA Replication; Female; Fluorescent Antibody Technique; Macrophage-1 Antigen; Male; Mice; Mice, Inbred C3H; Myelin Basic Protein; Schwann Cells; Sciatic Nerve; Thymidine; Tritium; Wallerian Degeneration

We examined the expression of mRNA encoding proteolipid protein (PLP), the major myelin protein in the CNS, in developing rat cerebrum, and in normal and degenerating optic nerves. PLP transcripts were initiated at two clusters of start sites that were separated by about 30 base pairs. During the peak of PLP mRNA expression in developing cerebrum, a higher proportion of PLP transcripts were initiated from the distal start site, furthest from the open reading frame, than in mature cerebrum. We enucleated one eye of immature rats to cause Wallerian degeneration in the optic nerve. In these degenerating optic nerves, the steady state levels of PLP mRNA fell markedly, and the proportion of distally initiated PLP transcripts declined to the same proportion found in normal adult nerves. Changes in myelin gene expression were not limited to PLP mRNA, as the steady-state levels of myelin basic protein (MBP) mRNA paralleled those of PLP mRNA in the developing cerebrum and in degenerating optic nerves. Thus, oligodendrocytes require axons to maintain their normal levels of PLP and MBP transcripts and the high proportion of distally initiated PLP transcripts that characterize early myelination.

Topics: Animals; Axons; Base Sequence; Blotting, Northern; Brain Chemistry; Densitometry; In Vitro Techniques; Molecular Sequence Data; Myelin Basic Protein; Oligonucleotides; Optic Nerve; Proteolipids; Rats; Rats, Sprague-Dawley; RNA, Messenger; Wallerian Degeneration

Myelin basic protein (MBP) processed by macrophages was reported to promote Schwann cell division during Wallerian degeneration. In the present study we have shown that there was no difference in the rate of Schwann cell proliferation between shiverer mice, which totally lack MBP, and control mice after nerve-transection. Furthermore, addition of autologous peritoneal macrophages in cultures led to enhanced thymidine uptake by Schwann cells, irrespective of the presence or absence of MBP. These results suggest that myelin components other than MBP play a role in Schwann cell proliferation induced by macrophages.

Topics: Animals; Cell Division; Cells, Cultured; Macrophages; Mice; Mice, Inbred C3H; Mice, Neurologic Mutants; Myelin Basic Protein; Schwann Cells; Sciatic Nerve; Thymidine; Wallerian Degeneration

Chronic relapsing experimental autoimmune encephalomyelitis is commonly seen in a number of species after a single injection of whole white matter in adjuvant but not after inoculation with myelin basic protein, the major encephalitogen of central myelin. In the present report on large groups of SJL mice, we describe a form of chronic relapsing experimental autoimmune encephalomyelitis with destructive lesions after a single inoculation of myelin basic protein in complete Freund's adjuvant. This condition was studied for up to 19 months postinoculation and was characterized by a relapsing-remitting or a chronic progressive course, usually with a prolonged latent period. Higher doses of 400 and 800 micrograms of myelin basic protein were more effective in inducing this condition than were lower doses of 100 and 200 micrograms. Large lesions were apparent in the white matter. These comprised widespread destruction and Wallerian degeneration with some demyelination towards the margins. Demyelination was an initial, albeit transient, event which was subsequently masked by nerve fiber destruction. Polymorphonuclear leukocytes were early and prominent components of the inflammatory infiltrate and together with macrophages appeared to be involved in the lysis of myelin and axons. Thus, despite the clinical similarities, these features contrast the model with the more purely demyelinative lesions of chronic relapsing experimental autoimmune encephalomyelitis in other species and multiple sclerosis in man.

Topics: Adjuvants, Immunologic; Animals; Demyelinating Diseases; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Inflammation; Macrophages; Mice; Mice, Inbred Strains; Multiple Sclerosis; Myelin Basic Protein; Neutrophils; Wallerian Degeneration

After transection of the mouse sciatic nerve, the sequence of events occurring in the distal degenerating segment was followed by the biochemical changes related to the cytoskeletal components and to the myelin protein markers. The components of the intermediate filaments and of the microtubules undergo early changes. Within 3 days, the neurofilament triplet and the peripherin disappear whereas many peptides bearing the antigenic determinant common to all classes of intermediate filaments accumulate. Several of them persist after 1 month. The tubulin pattern changes from a high level of microheterogeneity--reflecting mostly the axonal contribution--to a lower level displayed by the predominant Schwann cells. A decrease in the amount of the myelin markers is also observed. However, a month after transection, immunoreactive basic protein is still present in the degenerated segment homogenate.

Topics: Animals; Cytoskeleton; Electrophoresis, Polyacrylamide Gel; Intermediate Filament Proteins; Isoelectric Point; Membrane Glycoproteins; Mice; Mice, Inbred CBA; Molecular Weight; Myelin Basic Protein; Myelin Proteins; Nerve Degeneration; Nerve Tissue Proteins; Neurofilament Proteins; Peripherins; Sciatic Nerve; Tubulin; Vimentin; Wallerian Degeneration

Changes of myelin proteins in mouse sciatic nerves were studied comparing nerves degenerating in situ with nerves enclosed in millipore diffusion chambers which eliminate invasion of non-resident cells. Nerves kept in chambers showed nearly complete preservation of myelin sheaths with a very slow degradation of myelin proteins. Nerves degenerating in situ showed rapid myelin phagocytosis by macrophages with almost complete disappearance of myelin proteins after 28 days. These data elucidate the role of macrophages for removal of myelin proteins.

Topics: Animals; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Monocytes; Myelin Basic Protein; Myelin Sheath; Nerve Degeneration; Phagocytosis; Schwann Cells; Sciatic Nerve; Time Factors; Wallerian Degeneration

Topics: Animals; Antigens; Electrophoresis, Polyacrylamide Gel; Encephalomyelitis, Autoimmune, Experimental; Glycoproteins; Myelin Basic Protein; Myelin Proteins; Nerve Degeneration; Peripheral Nerves; Rabbits; Radioimmunoassay; Sciatic Nerve; Wallerian Degeneration