Myelin Splitting Research Articles

Effect of peripheral blood mononuclear cells on ischemia-reperfusion injury of sciatic nerve of adult male albino rat: histological, immunohistochemical, and ultrastructural study

ABSTRACT Ischemia/reperfusion (I/R) injury of sciatic nerve is a serious condition that results in nerve fiber degeneration, and reperfusion causes oxidative injury. Peripheral blood mononuclear cells (PBMNCs) have neuroregenerative power. This study was carried out to evaluate the potential ameliorative effect of PBMNCs on changes induced by I/R injury of the sciatic nerve. Fifty adult male albino rats were divided into donor and experimental groups that were subdivided into four groups: group I (control group), group II received 50 µL PBNMCs once intravenously via the tail vein, group III rubber tourniquet was placed around their Rt hind limb root for 2 hours to cause ischemia, group IV was subjected to limb ischemia as group III, then they were injected with 50 ul PBMNCs as group II before reperfusion. I/R injury showed disorganization of nerve fascicles with wide spaces in between nerve fibers. The mean area of collagen fibers, iNOS immunoexpression, and number of GFAP-positive Schwann cells of myelinated fibers showed a highly significant increase, while a highly significant reduction in the G-ratio and neurofilament immunoexpression was observed. Myelin splitting, invagination, evagination, and myelin figures were detected. PBMNC-treated group showed a marked improvement that was confirmed by histological, immunohistochemical, and ultrastructural findings.

Incidental Ultrastructural Findings in the Sural Nerve and Dorsal Root Ganglion of Aged Control Sprague Dawley Rats in a Nonclinical Carcinogenicity Study.

Xenobiotic-induced peripheral nerve damage is a growing concern. Identifying relative risks that a new drug may cause peripheral nerve injury over long periods of administration is gathering importance in the evaluation of animal models. Separating out age-related changes in peripheral nerves of rats caused by compression injury from drug-induced effects has been difficult. Biopsy of the sural nerve is utilized in humans for investigations of peripheral neuropathy, because it is largely removed from the effects of nerve compression. This study used transmission electron microscopy to identify incidental findings in the sural nerves and dorsal root ganglia of aged control rats over time. The goal was to establish a baseline understanding of the range of possible changes that could be noted in controls compared to rats treated with any new investigative drug. In this evaluation, most sural nerve fibers from aged control rats had few ultrastructural abnormalities of pathologic significance. However, glycogenosomes, polyglucosan bodies, swollen mitochondria, autolysosomes, split myelin, Schwann cell processes, and endoneural macrophages with phagocytosed debris (considered an indication of ongoing degenerative changes) were occasionally noted.

Time Course of Axon and Myelin Degeneration in Peripheral Nerves in Experimental Autoimmune Neuritis Rats

Experimental autoimmune neuritis (EAN) is an animal model for Guillain-Barré syndrome (GBS), which results in neurological symptoms and histopathological changes in peripheral nerves. In this model, the correlation between the progression of the disease and the histopathological changes is not clear. To further examine histopathological changes in peripheral nerves in EAN rats, sciatic nerves were sampled at onset (day 10), peak (day 16), and recovery (days 22 and 25) of neurological symptoms in P2(57-81)-peptide-administered rats. Axon and myelin degeneration was observed by light microscopy at onset, degeneration became severe at peak, and persisted at recovery. Densities of myelinated nerve fibers and myelin areas decreased from day 10 to a minimum on day 22. Slight axon and myelin degeneration, such as accumulation of vesicles in axons and focal myelin splitting and folding, was observed by transmission electron microscopy at onset; severe degeneration, such as axonal loss, myelin ovoid, and demyelination, increased at peak; and regenerative changes, such as remyelination and enlargement of Schwann cell cytoplasm, occurred at recovery. These results suggest that EAN rats have histopathological similarities to some types of GBS patients and that EAN rats are a useful model to understand the pathogenesis of GBS.

Empagliflozin Ameliorates Type 2 Diabetes-Induced Ultrastructural Remodeling of the Neurovascular Unit and Neuroglia in the Female db/db Mouse

Type 2 diabetes is associated with diabetic cognopathy. Anti-hyperglycemic sodium glucose transporter 2 (SGLT2) inhibitors have shown promise in reducing cognitive impairment in mice with type 2 diabetes mellitus. We recently described marked ultrastructural (US) remodeling of the neurovascular unit (NVU) in type 2 diabetic db/db female mice. Herein, we tested whether the SGLT-2 inhibitor, empagliflozin (EMPA), protects the NVU from abnormal remodeling in cortical gray and subcortical white matter. Ten-week-old female wild-type and db/db mice were divided into lean controls (CKC, n = 3), untreated db/db (DBC, n = 3), and EMPA-treated db/db (DBE, n = 3). Empagliflozin was added to mouse chow to deliver 10 mg kg−1 day−1 and fed for ten weeks, initiated at 10 weeks of age. Brains from 20-week-old mice were immediately immersion fixed for transmission electron microscopic study. Compared to CKC, DBC exhibited US abnormalities characterized by mural endothelial cell tight and adherens junction attenuation and/or loss, pericyte attenuation and/or loss, basement membrane thickening, glia astrocyte activation with detachment and retraction from mural cells, microglia cell activation with aberrant mitochondria, and oligodendrocyte–myelin splitting, disarray, and axonal collapse. We conclude that these abnormalities in the NVU were prevented in DBE. Empagliflozin may provide neuroprotection in the diabetic brain.

Histological, immunohistochemical and ultrastructural study of secondary compressed spinal cord injury in a rat model.

Spinal cord injury (SCI) is a life-disrupting condition in which the first few days are the most critical. Secondary conditions remain the main causes of death for people with SCI. The response of different cell types to SCI and their role at different times in the progression of secondary degeneration are not well understood. The aim of this study was to study the histopathological changes of compressed spinal cord injury (CSCI) in a rat model. Forty adult male Sprague-Dawley rats were divided into four groups. In group I, the rats were left without any surgical intervention (control). In group II, the rats were subjected to laminectomy without spinal cord compression (sham-operated). In group III, the rats were sacrificed one day after CSCI. In group IV, the rats were sacrificed seven days after CSCI. The light microscopy was employed to study the morphology using H&E, osmic acid staining and immunohistochemistry to detect glial fibrillary acidic protein (GFAP). The electron microscopy was applied for ultrastructure study. Histopathological examination of the posterior funiculus of the white matter revealed minute hemorrhages and localized necrotic areas on day 1, which transformed to areas of cavitation and fibrinoid necrosis surrounded by a demarcating rim of numerous astrocytes by day 7. The mean percentage of area of GFAP expression increased significantly by day 7. Osmic acid staining revealed swollen nerve fibers after one day, while numerous fibers had been lost by day 7. An ultrastructure study revealed swollen redundant thinned myelin and myelin splitting, as well as degeneration of axoplasm on day 1. On day 7, layers of the myelin sheath were folded and wrinkled with partial or complete demyelination areas. The myelin lamellae were disorganized and loose. The G-ratio was significantly greater on day 1 than day 7 after CSCI. In the rat model of CSCI details of the progressive spinal cord injury can be analyzed by morphological methods and may be helpful in the identification of the onset and type of clinical intervention.

Neuropathy in the spontaneously hypertensive rat: An electrophysiological and histological study.

Hypertension is identified as a risk factor for development of polyneuropathy. In this study we examined nerve conduction and morphological alteration of peripheral nerves in spontaneously hypertensive rats (SHR). Motor nerve conduction velocity (MNCV) in the sciatic-tibial nerve and sensory nerve conduction velocity (SNCV) in the sural nerve were measured. Pathological investigations included spinal cord, dorsal root ganglion, and hindlimb nerves in SHR and Wistar-Kyoto rats (WKY) aged 4-64 weeks. Blood pressure was significantly higher in SHR than WKY animals at 4 weeks and elevated further with aging. MNCV and SNCV were significantly slower in SHR compared with WKY after age 24 weeks. Prominent morphological changes in SHR nerves included axonal atrophy and myelin splitting. SHR also had endoneurial microangiopathy with reduplication of basement membrane. SHR showed slowed nerve conduction velocity and pathological abnormalities of hindlimb nerves. Sustained severe hypertension may cause axonal atrophy and endoneurial microangiopathy. Muscle Nerve 54: 756-762, 2016.

Novel pathologic findings in patients with Pelizaeus-Merzbacher disease

Pelizaeus-Merzbacher disease (PMD) is an X-linked inherited hypomyelinating disorder caused by mutations in the gene encoding proteolipid protein (PLP), the major structural protein in central nervous system (CNS) myelin. Prior to our study, whether hypomyelination in PMD was caused by demyelination, abnormally thin sheaths or failure to form myelin was unknown. In this study, we compared the microscopic pathology of myelin from brain tissue of 3 PMD patients with PLP1 duplications to that of a patient with a complete PLP1 deletion. Autopsy tissue procured from PMD patients was embedded in paraffin for immunocytochemistry and plastic for electron microscopy to obtain highresolution fiber pathology of cerebrum and corpus callosum. Through histological stains, immunocytochemistry and electron microscopy, our study illustrates unique pathologic findings between the two different types of mutations. Characteristic of the patient with a PLP1 deletion, myelin sheaths showed splitting and decompaction of myelin, confirming for the first time that myelin in PLP1 deletion patients is similar to that of rodent models with gene deletions. Myelin thickness and g-ratios of some fibers, in relation to axon diameter was abnormally thin, suggesting that oligodendrocytes remain metabolically functional and/or are attempting to make myelin. Many fibers showed swollen, progressive degenerative changes to axons in addition to the dissolution of myelin. All three duplication cases shared remarkable fiber pathology including swellings, constriction and/or transection and involution of myelin. Characteristic of PLP1 duplication patients, many axons showed segmental demyelination along their length. Still other axons had abnormally thick myelin sheaths, suggestive of continued myelination. Thus, each type of mutation exhibited unique pathology even though commonality to both mutations included involution of myelin, myelin balls and degeneration of axons. This pathology study describes findings unique to each mutation that suggests the mechanism causing fiber pathology is likewise heterogeneous.

Listeria monocytogenes spreads within the brain by actin-based intra-axonal migration.

Listeria monocytogenes rhombencephalitis is a severe progressive disease despite a swift intrathecal immune response. Based on previous observations, we hypothesized that the disease progresses by intra-axonal spread within the central nervous system. To test this hypothesis, neuroanatomical mapping of lesions, immunofluorescence analysis, and electron microscopy were performed on brains of ruminants with naturally occurring rhombencephalitis. In addition, infection assays were performed in bovine brain cell cultures. Mapping of lesions revealed a consistent pattern with a preferential affection of certain nuclear areas and white matter tracts, indicating that Listeria monocytogenes spreads intra-axonally within the brain along interneuronal connections. These results were supported by immunofluorescence and ultrastructural data localizing Listeria monocytogenes inside axons and dendrites associated with networks of fibrillary structures consistent with actin tails. In vitro infection assays confirmed that bacteria were moving within axon-like processes by employing their actin tail machinery. Remarkably, in vivo, neutrophils invaded the axonal space and the axon itself, apparently by moving between split myelin lamellae of intact myelin sheaths. This intra-axonal invasion of neutrophils was associated with various stages of axonal degeneration and bacterial phagocytosis. Paradoxically, the ensuing adaxonal microabscesses appeared to provide new bacterial replication sites, thus supporting further bacterial spread. In conclusion, intra-axonal bacterial migration and possibly also the innate immune response play an important role in the intracerebral spread of the agent and hence the progression of listeric rhombencephalitis.

Role of calorie restriction in alleviation of age-related morphological and biochemical changes in sciatic nerve.

Aging is associated with structural, functional and biochemical alterations in the nervous system. Calorie restriction (CR) was found to retard most physiological indices of aging. This work aimed to investigate the effect of CR on age-related changes in sciatic nerves. Thirty male albino rats aged 1 month were equally divided into three groups; Group I [control adult-ad libitum AL]: fed a regular diet and sacrificed at the age of 6 months, group II (aged-AL group): fed a regular diet AL and sacrificed at the age of 18 months, and group III (aged CR) fed a 40% calorie restricted diet and sacrificed at the age of 18 months. Rats were anesthetized and sciatic nerves were processed for light, electron microscope and morphometric studies. Oxidative stress in sciatic nerves was investigated by estimation of lipid perioxidation by product malondialdehyde (MDA) tissue level and antioxidant enzyme; superoxide dismutase activity (SOD). The aged (AL) sciatic nerves appeared disorganized, with thick perineurium and increased collagen fibers associated with decreased g-ratio. Abnormal myelin forms were seen as outfolded myelin loops, thin denuded myelin, splitting of myelin into myelin figures and interlamellar vacuoles. Schwann cells revealed vacuolated cytoplasm. There was also significant increase in MDA level and a significant decrease in SOD activity in comparison to control adult (AL). Apparent structural and histomorphological improvement were noticed after CR in aged rats. Aging caused structural and biochemical alterations in sciatic nerves with alleviating effect of calorie restriction on such effects.

Anti-GQ1b ganglioside positive Miller Fisher syndrome – evidence of paranodal pathology on nerve biopsy

Miller Fisher syndrome is a regional variant of Guillain-Barre syndrome with a characteristic clinical triad of ophthalmoplegia, areflexia and ataxia and occasionally distal limb sensory loss. 90% of patients have associated antibodies to the GQ1b ganglioside. The pathophysiology of antibody-mediated peripheral nerve impairment remains uncertain. This report includes the first description of a peripheral sensory nerve biopsy in Miller Fisher syndrome. A single case report is described of a 46 year old woman who presented with 2 weeks of distal glove and stocking sensory loss to both deep and superficial sensory modalities, areflexia and weight loss. This was followed by rapid onset of ataxia, ophthalmoplegia, and bulbar impairment. Peripheral neurophysiology showed reduced sensory nerve amplitudes with preserved conduction velocities in keeping with an axonal pattern of impairment. Clinical concerns of a systemic inflammatory disorder led to a diagnostic peripheral nerve biopsy from the sensory branch of the radial nerve. However she subsequently made a complete recovery over 5 weeks. Combinatorial glycoarrays confirmed restricted serum binding for GQ1b in acute serum which later resolved in a convalescent sample. The nerve biopsy showed lengthening of nodes of Ranvier, myelin splitting and macrophage internodal axonal invasion without any features of demyelination. The pathological features were strikingly similar to those found in acute motor axonal neuropathy and indicate the region of the node of Ranvier to be a primary focus of GQ1b induced damage in Miller Fisher syndrome, at least in this particular overlap syndrome with prominent sensory nerve involvement.

Neuroprotective Effect of Cerebrolysin on Diabetic Neuropathy: A Study on Male Rats

Objective: Diabetes mellitus with 10% prevalence in human population leads to disorders of peripheral nervous system in many affected patients. It causes various polyneuropathies in which nerve conductionvelocity decreases. The aim of this study was to investigate the effect of cerebrolysinon the treatment of neural injuries resulted from hyperglycemia. Method: Diabetes was induced in male rats weighing 250 ± 25 gr by intraperitoneal injection of 65 mg/kg streptozocin (STZ). Six weeks after STZ injection and appearance of neuropathy in diabetic rats, animals were divided into four groups: experimental, vehicle, diabetic and control. The experimental and vehicle groups received respectively single dose of 5 mg/kg day-1 cerebrolysinand saline intraperitoneally for two weeks. At the end, in order to find the efficacy of cerebrolysin, all groups underwent behavioral and electrophysiological tests as well as histological investigation. Results: Metabolic parameters in different groups showed inefficacy of cerebrolysin in the treatment of metabolic disorders of diabetes. However, electrophysiological investigations showed efficacy of cerebrolysin in the treatment of diabetic neuropathy in rats. Moreover, investigation on morphologic structure of sciatic nerve was evident of the return of axon degenerative changes and myelin splitting in nerve fibers in cerebrolysin-received group. The results of behavioral studies showed increase in recovery in cerebrolysin group. Conclusion: According to the results, treatment of diabetic neuropathy with daily injection of 5 mg/kg cerebrolysin for two weeks improves rats’ condition.

What T-cell receptors can tell us about neurologic disease

Chronic demyelinating polyradiculoneuropathy (CIDP) is an acquired, potentially crippling sensory-motor polyneuropathy of variable course.1 It is likely autoimmune because sural nerve histopathology reveals infiltrating CD68+ macrophages, endoneurial and epineurial CD3+ T cells with immunoglobulin G, immunoglobulin M, and complement deposits on myelin sheaths.1 Macrophages penetrate the basal lamina of Schwann cells and split myelin lamellae, a process referred to as “macrophage-mediated demyelination.” Nerve injury ensues due to a combination of antibody-, complement-, and macrophage-dependent demyelination. The majority of patients therefore respond to corticosteroids, plasmapheresis, or IV immunoglobulin (IVIg). The Immune Globulin IV CIDP Efficacy (ICE) study, the largest therapeutic trial in CIDP, convincingly demonstrated short- and long-term benefits of IVIg treatment.2 Response to plasmapheresis or IVIg is consistent with humoral and Fc receptor–mediated cellular effector mechanisms. In this issue of Neurology ®, Schneider-Hohendorf et al.3 present evidence that CD8+ cytotoxic T cells contribute to the pathogenesis of CIDP: nerve-infiltrating CD8+ T cells are clonally expanded, with expanded T-cell clones also detectable in blood. In one especially informative patient, expanded T-cell receptor (TCR) β-chain variable (TCRVβ) 5.1+ T cells were enriched in sural nerve compared to blood. To interpret and understand these results, one needs to consider the methodology used in the study. CD8+ (cytotoxic) T cells typically recognize a short …

DIFFERENTIAL DIAGNOSIS OF ORGANIC ACIDEMIA: CLINICAL AND NEUROIMAGING FINDINGS

Clinical differential DiagnosisThe organic acidemias are important in the differential diagnosis of metabolic and neurologic derangement in the neonate and of new-onset neurologic signs in the older child. A-Organic aciduriaSeveral disorders, not classified as primary disorders of organic acid metabolism, have a characteristic urinary organic acid profile that suggests the appropriate diagnosis. • Mevalonicaciduria, a disorder of cholesterol biosynthesis, shows mevalonic acid in the urine. • Glutaricacidemia type II, a disorder of fatty acid oxidation, has multiple organic acids in abnormal concentration in urine. These organic acids include ethylmalonic acid, glutaric acid, dicarboxylic acids, and glycine conjugates of medium chain dicarboxylic acids. • The fatty acylCoA-glycine conjugates that signal incomplete fatty acid oxidation and serves as signals to the diagnosis of MCAD defeciency and other disorders of fatty acid oxidation and transport. • Biotinidase deficiency, a disorder of biotin recycling, results in the urinary excretion of several unusual organic acids, including 3-hydroxy-isovaleric, 3-hydroxypropionic, 3-hydroxybutyric acids, and acetoacetate. Propionyl glycine may also be seen. • Mitochondrial diseases with disordered oxidative phosphorylation often demonstrate the presence of abnormal organic acids in the urine. B-AcidosisNon-genetic conditions, such as shock, sepsis, DKA, liver and kidney failure, thiamine deficiency, RTA, some drug intoxication cause acidosis- genetic conditions are include: inherited metabolic disorders of lactate and pyruvate metabolism and oxidative phosphorylation, disorders of the Krebs cycle such as fumarase deficiency. C-HyperammonemiaDisorders of the urea cycle and the hyperammonemia-hypoglycemia syndrome.Neuroimaging• A variety of MRI abnormalities have been described in the organic academia, including distinctive basal ganglia lesions in glutaricacidemia type I (GA I), white matter changes in maple syrup urine disease (MSUD), and abnormalities of the globus pallidus in methylmalonic acidemia. Macrocephaly is common in GA I. • Some differential agnosis of MRI findings in organic academia is consist of: HIE, mucopolysacaridosis, middle fossa arachnoid cyst, leighdisease, hexachlorophene toxicity in neonates, myelin splitting disorders. • Some organic aciduria such as L-2-Hydroxyglutaricaciduria may suggest leukodystrophy in MRI.

Acrolein induces myelin damage in mammalian spinal cord

Myelin damage can lead to the loss of axonal conduction and paralysis in multiple sclerosis and spinal cord injury. Here, we show that acrolein, a lipid peroxidation product, can cause significant myelin damage in isolated guinea pig spinal cord segments. Acrolein-mediated myelin damage is particularly conspicuous in the paranodal region in both a calcium dependent (nodal lengthening) and a calcium-independent manner (paranodal myelin splitting). In addition, paranodal protein complexes can dissociate with acrolein incubation. Degraded myelin basic protein is also detected at the paranodal region. Acrolein-induced exposure and redistribution of paranodal potassium channels and the resulting axonal conduction failure can be partially reversed by 4-AP, a potassium channel blocker. From this data, it is clear that acrolein is capable of inflicting myelin damage as well as axonal degeneration, and may represent an important factor in the pathogenesis in multiple sclerosis and spinal cord injury.

Effects of Melatonin in Prevention of Neuropathy in STZ-Induced Diabetic Rats

Problem statement: Diabetes mellitus occurs mainly with chronic polyneuropathy, and oxidative stress plays an important role in emergence of most neurologic and behavioral changes in diabetic patients. Many studies have focused on the beneficial effects of various antioxidants such as melatonin on diabetic neuropathy. The aim of this study is to evaluate the effect of melatonin in prevention of neuropathy in Streptozotocin-induced diabetic rats. After prescribing Streptozotocin (STZ), treatment rats received melatonin (10 mg kg day-1) or DMSO for a period of 6 weeks. Approach: At the end of the sixth week, non diabetic control group, diabetic control group (sham) and treated rats were examined by thermal pain response tests (hot plate and tail flick). The horizontal and vertical activities of rats were measured in an open field test. After that, Motor Nerve Conduction Velocity (MNCV) of sciatic-tibial nerve recorded. Also, to study morphological alterations resulting from diabetic neuropathy of sciatic nerve, Myelinated Fiber Diameter (MFD), Axon Diameter (AD) and Myelin Sheath Diameter (MSD) were evaluated by light microscope. Results: According to hot plate results, response time to thermal pain at the end of sixth week in sham group showed a significant decrease in comparison with the control group (p<0.01). In hot plate test, although melatonin approximated to the response time to control group, the significant difference was not observed among melatonin receivers and other groups. In the open field test, Total Distance Moved (TDM) and mobility duration showed significant decrease in sham and DMSO groups in comparison to the control and melatonin groups. Diabetic rats treated with melatonin showed significant increase in MNCV compared to sham and DMSO groups (p<0.05). In morphological study, pretreatment with Melatonin significantly reversed sciatic nerve diameters (MFD, AD, and MSD) reduction in diabetic rats. Electron microscopy showed myelin splitting and myelin sheath infolding in diabetic control group compare to non diabetic group. Conclusion: This study showed that melatonin can decrease the destructive progress of diabetes and causes neuroprotection against damages resulting from STZ-induced hyperglycemia.

Structural abnormalities in the cortex of the rTg4510 mouse model of tauopathy: a light and electron microscopy study

rTg4510 transgenic (TG) mice overexpress mutant (P301L) human tau protein. We have compared the dorsal premotor cortex of TG mice versus non-transgenic (NT) mice at 4, 9, and 13months of age, using light (LM) and electron microscopy (EM). LM assessment shows that cortical thickness in TG mice is reduced by almost 50% from 4 to 13months of age, while at the same time layer I thickness is reduced by 80%, with most of the cortical thinning occurring between 4 and 9months. In TG mice, spherical, empty vacuoles, up to 60μm in diameter, become increasingly abundant with age and by 9months, pyramidal and non-pyramidal neurons with large intracellular tangles of tau protein are common throughout the cortex. These tangles occur in the perikarya; we have not observed them entering into cellular processes, nor have we observed ghost tangles in the intercellular matrix. In TG mice, nerve fiber pathology is widespread by 13months, and split myelin sheaths, ballooned sheaths, and swollen axons containing mitochondrial aggregations are all common. Astrocytes become increasingly filled with glial filaments as TG mice age, and microglial cells almost always contain phagocytic inclusions. However, no glial cells are seen to contain tau in their cytoplasm. These observations add to the base of knowledge available on this commonly employed model of tauopathy.

Adult-onset leukodystrophy with axonal spheroids

We report two cases of adult-onset leukodystrophy with axonal spheroids in a brother and sister, one with an autopsy, the other with a cerebral biopsy. Symptoms developed at 24 and 33 years. The sister died within four years, her brother in 2 and a half. Her brain showed a large lesion in the frontal centrum semiovale extending through the corpus callosum and shading off into the normal appearing U-fibers. With routine stains axonal spheroids were most easily detected in the mildly affected regions, although stains for neurofilaments showed that they were actually more numerous, though more irregular in shape, where the lesions were more severe. Axons and myelin appeared equally affected. Pigmented cells were absent. Electron microscopy on the cerebral biopsy showed that the spheroids contained neurofilaments and neurotubules, mitochondria and degenerate mitochondria, with a suggestion of polarity such that neurofilaments were more common in the proximal part of spheroids. Many other axons were found whose size was only slightly increased although they were filled with sacs of abnormal granular material which were probably derived from mitochondria. The anterior commissure and the corticospinal tracts in the spinal cord of the sister showed fiber loss and large vacuoles within which occasional intact axons could be identified. This was considered to represent intramyelin edema. In the cerebral biopsy one vacuole arising from myelin splitting was found in confirmation of this hypothesis. We conclude that both myelin and axons are damaged, although the lesions of myelin are conditioned by axonal abnormalies in this genetic disease.

Glutamate Excitotoxicity Inflicts Paranodal Myelin Splitting and Retraction

Paranodal myelin damage is observed in white matter injury. However the culprit for such damage remains unknown. By coherent anti-Stokes Raman scattering imaging of myelin sheath in fresh tissues with sub-micron resolution, we observed significant paranodal myelin splitting and retraction following glutamate application both ex vivo and in vivo. Multimodal multiphoton imaging further showed that glutamate application broke axo-glial junctions and exposed juxtaparanodal K+ channels, resulting in axonal conduction deficit that was demonstrated by compound action potential measurements. The use of 4-aminopyridine, a broad-spectrum K+ channel blocker, effectively recovered both the amplitude and width of compound action potentials. Using CARS imaging as a quantitative readout of nodal length to diameter ratio, the same kind of paranodal myelin retraction was observed with applications of Ca2+ ionophore A23187. Moreover, exclusion of Ca2+ from the medium or application of calpain inhibitor abolished paranodal myelin retraction during glutamate exposure. Examinations of glutamate receptor agonists and antagonists further showed that the paranodal myelin damage was mediated by NMDA and kainate receptors. These results suggest that an increased level of glutamate in diseased white matter could impair paranodal myelin through receptor-mediated Ca2+ overloading and subsequent calpain activation.

Experimental observation and theoretical analysis of Raman resonance-enhanced photodamage in coherent anti-Stokes Raman scattering microscopy

The photodamage in coherent anti-Stokes Raman scattering (CARS) imaging of spinal tissues is featured by plasma-induced myelin splitting and shockwaves. When the excitation is tuned on resonance with the symmetric CH2 stretch vibration, the average point-scanning time to cause the photodamage is reduced by half. Similar Raman resonance-enhanced photodamage is also observed for a polymer film. The light-matter energy transfer in coherent Raman processes with both plane waves and focused excitation beams is analyzed to interpret this phenomenon. Our calculation indicates that at Raman resonance, a significant vibrational absorption in the material can be stimulated by the concomitant Raman gain and Raman loss processes due to high incident-field intensities under a tight-focusing condition. As a result, while the nonlinear damage induced by multiphoton absorption can be diminished in CARS microscopy owing to the use of near-infrared picosecond pulses, the coherent Raman-induced vibrational pumping is able to enhance the photodamage by assisting plasma generation in the material.

The effects of a dominant connexin32 mutant in myelinating Schwann cells

Mutations in GJB1, the gene encoding the gap junction protein connexin32 (Cx32), cause X-linked Charcot–Marie–Tooth disease, an inherited demyelinating peripheral neuropathy. We generated transgenic mice that express the R142W mutation in myelinating Schwann cells. The R142W mutant protein was aberrantly localized to the Golgi, indicating that it does not traffic properly, but the molecular organization of the myelin sheath, including the localization of Cx29, another connexin expressed by myelinating Schwann cells, was not disrupted. In a wild type background, this mutation dramatically decreased the level of wild type mouse Cx32 in immunoblots of sciatic nerve and caused demyelination. The expression of wild type human Cx32 with the same transgenic construct had different effects—increased amounts of Cx32, normal localization of Cx32 at nodes and incisures, and split myelin sheaths. Thus, the R142W mutant protein has dominant effects that are distinct from overexpression.