Myelin Basic Protein Immunostaining Research Articles

Ongoing effects of preterm birth on the dopaminergic and noradrenergic pathways in the frontal cortex and hippocampus of guinea pigs.

Children born preterm have an increased likelihood of developing neurobehavioral disorders such as attention-deficit hyperactivity disorder (ADHD) and anxiety. These disorders have a sex bias, with males having a higher incidence of ADHD, whereas anxiety disorder tends to be more prevalent in females. Both disorders are underpinned by imbalances to key neurotransmitter systems, with dopamine and noradrenaline in particular having major roles in attention regulation and stress modulation. Preterm birth disturbances to neurodevelopment may affect this neurotransmission in a sexually dimorphic manner. Time-mated guinea pig dams were allocated to deliver by preterm induction of labor (gestational age 62 [GA62]) or spontaneously at term (GA69). The resultant offspring were randomized to endpoints as neonates (24h after term-equivalence age) or juveniles (corrected postnatal day 40, childhood equivalence). Relative mRNA expressions of key dopamine and noradrenaline pathway genes were examined in the frontal cortex and hippocampus and quantified with real-time PCR. Myelin basic protein and neuronal nuclei immunostaining were performed to characterize the impact of preterm birth. Within the frontal cortex, there were persisting reductions in the expression of dopaminergic pathway components that occurred in preterm males only. Conversely, preterm-born females had increased expression of key noradrenergic receptors and a reduction of the noradrenergic transporter within the hippocampus. This study demonstrated that preterm birth results in major changes in dopaminergic and noradrenergic receptor, transporter, and synthesis enzyme gene expression in a sex- and region-based manner that may contribute to the sex differences in susceptibility to neurobehavioral disorders. These findings highlight the need for the development of sex-based treatments for improving these conditions.

The restoration of hippocampal nerve de-myelination by methylcobalamin relates with the enzymatic regulation of homocysteine level in a rat model of moderate grade hepatic encephalopathy.

This article describes how methylcobalamin (MeCbl) restores nerve myelination in a moderate- grade hepatic encephalopathy (MoHE) model of ammonia neurotoxicity. The comparative profiles of myelin basic protein (MBP), homocysteine (Hcy) and methionine synthase (MS: a MeCbl- dependent enzyme) activity versus nerve myelination status were studied in the hippocampus of the control, the MoHE (developed by administering 100 mg/kg bw thioacetamide i.p. for 10 days) and the MoHE rats treated with MeCbl (500 µg/kg BW i.p.) for 7 days. Compared to those of control rats, the hippocampal CA1 and CA3 regions of the MoHE rats showed significantly lower myelinated areas and MBP immunostaining. This coincided with the deranged myelin layering in TEM images, decreased MBP protein and its transcript levels in hippocampus of MoHE rats. However, all these parameters recovered to normal levels after MeCbl treatment. MeCbl is a cofactor of MS that catalyzes the conversion of Hcy to methionine as a feeder step of methylation reactions. We observed significantly increased serum and hippocampal Hcy levels in MoHE rats, however, these levels were restored to control values with a concordant activation of MS due to MeCbl treatment. A significant recovery in neurobehavioral impairments in the MoHE rats due to MeCbl treatment was also observed. These findings suggest that MoHE pathogenesis is associated with deranged nerve myelination in the hippocampus and that MeCbl treatment is able to restore it mainly by activating MS, a MeCbl-dependent Hcy-metabolizing enzyme.

A scaffold containing zinc oxide for Schwann cell-mediated axon growth

Objective. Schwann cells (SCs) transplanted in damaged nervous tissue promote axon growth, which may support the recovery of function lost after injury. However, SC transplant-mediated axon growth is often limited and lacks direction. Approach. We have developed a zinc oxide (ZnO) containing fibrous scaffold consisting of aligned fibers of polycaprolactone (PCL) with embedded ZnO nanoparticles as a biodegradable, bifunctional scaffold for promoting and guiding axon growth. This scaffold has bifunctional properties wherein zinc is released providing bioactivity and ZnO has well-known piezoelectric properties where piezoelectric materials generate electrical activity in response to minute deformations. In this study, SC growth, SC-mediated axon extension, and the presence of myelin basic protein (MBP), as an indicator of myelination, were evaluated on the scaffolds containing varying concentrations of ZnO in vitro. SCs and dorsal root ganglion (DRG) neurons were cultured, either alone or in co-culture, on the scaffolds. Main results. Findings demonstrated that scaffolds with 1 wt.% ZnO promoted the greatest SC growth and SC-mediated axon extension. The presence of brain-derived neurotrophic factor (BDNF) was also determined. BDNF increased in co-cultures for all scaffolds as compared to SCs or DRGs cultured alone on all scaffolds. For co-cultures, cells on scaffolds with low levels of ZnO (0.5 wt.% ZnO) had the highest amount of BDNF as compared to cells on higher ZnO-containing scaffolds (1 and 2 wt.%). MBP immunostaining was only detected in co-cultures on PCL control scaffolds (without ZnO). Significance. The results of this study demonstrate the potential of the ZnO-containing scaffolds for SC-mediated axon growth and its potential for use in nervous tissue repair.

Deciphering the Global Proteomic Profile Involved in Methylmercury-Induced Cerebellar Neurodegeneration and Motor Dysfunction in Adult Rats.

Mercury is a ubiquitous pollutant in the environment with potential neurotoxic effects. Several populations are susceptible to mercurial exposure, especially methylmercury (MeHg) at low doses for long periods through food consumption. Given this, the present work aimed to assess the effects of long-term MeHg exposure on the cerebellum of rats from a translational perspective using a representative dose, assessing molecular, biochemical, morphological, and behavioral parameters. The model was produced by administering 40 µg/kg of MeHg for 60 days to adult male Wistar rats by oral gavage. As a result of this exposure, the animals presented motor deficits in open field and rotarod tests which were associated with an increase in total mercury content in cerebellar parenchyma, a reduction in antioxidant competence against peroxyl radicals, and increased nitrite and lipid peroxidation levels. The proteomic approach showed 317 modulated proteins. Such findings were associated with reductions in mature neuron and Purkinje cell densities and glial fibrillary acidic protein immunostained areas and increased microglial density. In addition, decreases in myelin basic protein and synaptophysin immunostaining were also observed. The results thus provided new evidence of the mechanisms underlying complex MeHg-induced neurodegeneration, especially the proteins underlying the biochemical and morphological features associated with motor dysfunction.

Low-field magnetic stimulation improved cuprizone-induced depression-like symptoms and demyelination in female mice.

Depression is a common and disabling comorbidity of multiple sclerosis (MS), with currently no clear guidelines for treatment. Low-field magnetic stimulation (LFMS), a novel non-invasive neuromodulation intervention, has been previously demonstrated to rapidly alleviate mood disorders. The aim of the present study was to investigate the effects of LFMS on depression-like behaviors and demyelination in a well-established mouse model of MS. C57BL/6 female mice were fed a 0.2% cuprizone (CPZ) diet for 3 or 6 weeks to induce acute demyelination. During this time, the mice were treated with either sham or LFMS for 20 min/day, 5 days/week. After 3 or 6 weeks of treatment, behavior was assessed with the open field task, Y-maze and the forced swim test. The prefrontal cortex and hippocampus were then collected to perform immunohistochemistry and western blot analysis to verify myelination status. The CPZ diet did not cause significant locomotor deficits; however, working memory, measured using the Y maze, depression-like behavior and adaptive learning, assayed using the forced swim test, were significantly impaired in these animals. LFMS treatment demonstrated a significant antidepressant-like effect and markedly attenuated the CPZ-induced demyelination in the prefrontal cortex after 3- and 6-weeks of treatment, as observed by changes in myelin basic protein immunostaining and western blot analysis. Therefore, the results of the present study indicated that LFMS may be a promising therapy for demyelinating diseases due to the improvement of depressive symptoms via regulation of myelination in cortical areas.

Adipose-Derived Mesenchymal Stem Cells From a Hypoxic Culture Improve Neuronal Differentiation and Nerve Repair.

Hypoxic expansion has been demonstrated to enhance in vitro neuronal differentiation of bone-marrow derived mesenchymal stem cells (BMSCs). Whether adipose-derived mesenchymal stem cells (ADSCs) increase their neuronal differentiation potential following hypoxic expansion has been examined in the study. Real-time quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were employed to detect the expression of neuronal markers and compare the differentiation efficiency of hypoxic and normoxic ADSCs. A sciatic nerve injury animal model was used to analyze the gastrocnemius muscle weights as the outcomes of hypoxic and normoxic ADSC treatments, and sections of the regenerated nerve fibers taken from the conduits were analyzed by histological staining and immunohistochemical staining. Comparisons of the treatment effects of ADSCs and BMSCs following hypoxic expansion were also conducted in vitro and in vivo. Hypoxic expansion prior to the differentiation procedure promoted the expression of the neuronal markers in ADSC differentiated neuron-like cells. Moreover, the conduit connecting the sciatic nerve gap injected with hypoxic ADSCs showed the highest recovery rate of the gastrocnemius muscle weights in the animal model, suggesting a conceivable treatment for hypoxic ADSCs. The percentages of the regenerated myelinated fibers from the hypoxic ADSCs detected by toluidine blue staining and myelin basic protein (MBP) immunostaining were higher than those of the normoxic ones. On the other hand, hypoxic expansion increased the neuronal differentiation potential of ADSCs compared with that of the hypoxic BMSCs in vitro. The outcomes of animals treated with hypoxic ADSCs and hypoxic BMSCs showed similar results, confirming that hypoxic expansion enhances the neuronal differentiation potential of ADSCs in vitro and improves in vivo therapeutic potential.

Noninvasive Assessment of Neurodevelopmental Disorders after In Utero Irradiation in Mice: An In Vivo Anatomical and Diffusion MRI Study.

In utero exposure to ionizing radiation can lead to cerebral alterations during adulthood. Using anatomical magnetic resonance imaging (MRI), it is possible to assess radiation-induced structural brain damage noninvasively. However, little is currently known about microstructure alterations in brain tissue. Therefore, the goal of this study was to establish, based on an original and robust pipeline of MRI image analysis, whether the long-term effects of in utero radiation exposure on brain tissue microstructure could be detected noninvasively. Pregnant C57BL/6N mice received a single dose of 1 Gy on gestation day 14.5, which led to behavioral impairments in adults. At 3 months old, in vivo MRI data were acquired from in utero irradiated and nonirradiated male mice. An MRI protocol was designed to assess the effects of radiation on the parameters of brain volume, non-Gaussian diffusion (ADC0, kurtosis and signature index) and anisotropic diffusion (fractional anisotropy and mean, axial, radial diffusivities and anisotropic signature index) in 10 key cerebral structures defined using an in-house atlas of the mouse brain. Based on the relative amplitude of these anatomical and microstructural changes, maps of the radiosensitivity of the brain to in utero irradiation were created. We observed microcephaly in irradiated mice with noticeably larger volume changes in the cortex and the corpus callosum. We also observed significantly lower ADC0, anisotropy fraction (sFA), radial diffusivity (sRD), as well as signature index (S-index and SI3) values, which are original markers sensitive to tissue microstructure alterations. All these changes together are in favor of a decreased cellular "imprint" and in some regions a reduced density in myelinated axons. A reduction in the number and complexity of myelinated axons was further revealed by myelin basic protein immunostaining. Combining anatomical and diffusion MRI is a promising approach to noninvasively investigate the radiosensitivity of local brain areas in adult mice after in utero irradiation in terms of microstructure.

Effects of Lipopolysaccharide on Oligodendrocyte Differentiation at Different Developmental Stages: an In Vitro Study.

BackgroundLipopolysaccharide (LPS) exerts cytotoxic effects on brain cells, especially on those belonging to the oligodendrocyte lineage, in preterm infants. The susceptibility of oligodendrocyte lineage cells to LPS-induced inflammation is dependent on the developmental stage. This study aimed to investigate the effect of LPS on oligodendrocyte lineage cells at different developmental stages in a microglial cell and oligodendrocyte co-culture model.MethodsThe primary cultures of oligodendrocytes and microglia cells were prepared from the forebrains of 2-day-old Sprague–Dawley rats. The oligodendrocyte progenitor cells (OPCs) co-cultured with microglial cells were treated with 0 (control), 0.01, 0.1, and 1 µg/mL LPS at the D3 stage to determine the dose of LPS that impairs oligodendrocyte differentiation. The co-culture was treated with 0.01 µg/mL LPS, which was the lowest dose that did not impair oligodendrocyte differentiation, at the developmental stages D1 (early LPS group), D3 (late LPS group), or D1 and D3 (double LPS group). On day 7 of differentiation, oligodendrocytes were subjected to neural glial antigen 2 (NG2) and myelin basic protein (MBP) immunostaining to examine the number of OPCs and mature oligodendrocytes, respectively.ResultsLPS dose-dependently decreased the proportion of mature oligodendrocytes (MBP+ cells) relative to the total number of cells. The number of MBP+ cells in the early LPS group was significantly lower than that in the late LPS group. Compared with those in the control group, the MBP+ cell numbers were significantly lower and the NG2+ cell numbers were significantly higher in the double LPS group, which exhibited impaired oligodendrocyte lineage cell development, on day 7 of differentiation.ConclusionRepetitive LPS stimulation during development significantly inhibited brain cell development by impairing oligodendrocyte differentiation. In contrast, brain cell development was not affected in the late LPS group. These findings suggest that inflammation at the early developmental stage of oligodendrocytes increases the susceptibility of the preterm brain to inflammation-induced injury.

Effects of prenatal stress on behavioural and neurodevelopmental outcomes are altered by maternal separation in the neonatal period

BackgroundChronic psychosocial stress during pregnancy and/or after birth, and the associated elevation in cortisol, is linked with the onset of behavioural disorders in childhood. Previously, prenatal stress has been shown to reduce neurosteroid pathways in the fetus and the levels of the neurosteroid and GABAA receptor agonist, allopregnanolone. In late gestation, elevated levels of GABAergic activity increases inhibitory tone and protects against excessive excitation. These levels of allopregnanolone may also contribute to promoting myelination, thus stress-induced suppression of protective neurosteroid levels may disrupt neurodevelopmental processes and can result in reduced myelination. The objective of this study was to examine whether prenatal and postnatal stress reduces levels of inhibitory pathways to result in behavioural, myelin, and GABAergic/glutamatergic pathway deficits in the hippocampus at a postnatal time point in the guinea pig equivalent to childhood in humans. MethodsPregnant guinea pig dams were exposed to prenatal stress (PRE) with strobe light exposure for 2 h/day on gestational age (GA) 50, 55, 60 and 65 (term is ∼GA70), with postnatal stress (POST) caused by maternal separation for 2 h/day from postnatal day (PND) 1–7), or a double-hit of both stressors (PRE + POST). Control dams and offspring groups (CON) were handled at the same time each day without causing stress. Behavioural outcomes were assessed using open field and elevated plus maze testing on PND27. After euthanasia on PND30, plasma samples were collected for steroid quantification of cortisol, allopregnanolone and progesterone by ELISA. Hippocampal samples were collected to assess markers of oligodendrocyte development and mature cells by myelin basic protein (MBP) immunostaining and GABAergic and glutamatergic pathway component gene expression by real time PCR. ResultsMale guinea pig offspring exposed to prenatal stress exhibited hyperactive-like behaviour at childhood equivalence, while female offspring displayed anxious-like behaviour, to a lesser extent. In both sexes, MBP immunostaining was significantly decreased in the hippocampal region following prenatal stress, despite normal levels of MBP mRNA, which suggests a disruption to the MBP protein translation pathway. Many components of the GABAergic and glutamatergic pathways were disrupted following prenatal stress, notably GABAA receptor subunits, GABA production and uptake, glutamate ionotropic and metabotropic receptor subunits and glutamate transport. Following prenatal + postnatal stress, many of the behavioural and neurodevelopmental deficits were improved compared to the prenatal stress only group. ConclusionWe conclude that prenatal stress disrupts GABAergic and glutamatergic pathways that may contribute to reduced myelination and subsequent behavioural deficits in the offspring. The deficits seen following prenatal stress are ameliorated when paired with subsequent postnatal stress, which highlights the early postnatal period as an important treatment window.

Eff ects of hyperbaric oxygen on NLRP3 infl ammasome activation in the brain aft er carbon monoxide poisoning

Neuroinflammation plays an important role in brain damage after acute carbon monoxide poisoning (ACOP). The nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing (NLRP) 3 inflammasome triggers the activation of inflammatory caspases and maturation of interleukin (IL)-1β and -18, and has been linked to various human autoinflammatory and autoimmune diseases. In this study we investigated the effects of hyperbaric oxygen (HBO2) on NLRP3 inflammasome activation after ACOP. Mice were randomly divided into four groups: sham group (exposure to normobaric air – i.e., 21% O2 at 1 atmosphere absolute); HBO2-only group; CO + normobaric air group; and CO + HBO2 group. Cognitive function was evaluated with the Morris water maze; myelin injury was assessed by Fluoro-Myelin GreenTM fluorescent myelin staining and myelin basic protein (MBP) immunostaining; and mRNA and protein levels of NLRP3 inflammasome complex proteins were measured by quantitative real-time PCR and Western blot, respectively. Additionally, serum and brain levels of IL-1β and -18 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were determined by enzyme-linked immunosorbent assay. It was found that HBO2 improved learning and memory, and alleviated myelin injury in mice subjected to acute CO exposure. Furthermore, HBO2 decreased NLRP3, absent in melanoma 2 (AIM2), caspase-1, and apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain mRNA and protein levels, and reduced brain and serum concentrations of IL-1β and -18 and NADPH oxidase. These results indicate that HBO2 suppresses the inflammatory response after ACOP by blocking NLRP3 inflammasome activation, thereby alleviating cognitive deficits.

Sevoflurane Post-Conditioning Ameliorates Neuronal Deficits and Axon Demyelination After Neonatal Hypoxic Ischemic Brain Injury: Role of Microglia/Macrophage.

Microglia/macrophages have been identified to be highly polarized after ischemia. Interestingly, the polarization of these microglia/macrophages varies immensely under differing disease conditions. Post-conditioning using sevoflurane, a volatile anesthetic, could provide long-term neuroprotection to neonatal rats after hypoxic-ischemic brain injury (HIBI). Thus, the current study aimed at investigating the effects of sevoflurane post-conditioning (SPC) on microglia/macrophage polarization after HIBI induction in neonatal rats. Additionally, we aimed at identifying the underpinning mechanisms specifically related to autophagy and lysosomal protease enzyme, cathepsin B. To develop a HIBI model, 7-day-old Sprague-Dawley rats underwent left common carotid artery ligation followed by 2h of hypoxia. The role of microglia/macrophages in the neuroprotection conferred by SPC was examined by left-side intra-cerebroventricular injection with adenovirus vector carrying catB-GFP or rapamycin. The number of interleukin (IL)-1β+ cells, cathepsin B+ cells, light chain 3B positive (LC3B+) cells among ionized calcium binding adaptor molecule 1(Iba1+)cells to investigate microglia polarization, neuronal apoptosis to assess neuronal death in the acute phase were tested at 24h after HIBI. Behavioral tests including suspension test, Morris water maze tests were performed to investigate the long-term effects of SPC, at 21 to 34days post HIBI. Nissl staining and myelin basic protein (MBP) immunostaining to assess the long-term neuronal and myelin damage were performed at 34days after HIBI. Based on the obtained results post HIBI, we observed the cells that were positive for IL-1β, cathepsin B, and LC3B among Iba1 positive cell population in the hippocampus were significantly decreased after SPC treatment. SPC significantly attenuated the HIBI-induced increase in neuronal apoptosis, improved long-term cognitive function, and attenuated HI-induced decrease of Nissl-positive cells and MBP expression. However, these trends were reversed by injection of adenovirus vector carrying catB-GFP and rapamycin. SPC attenuated microglia polarization towards neurotoxic phenotypes, alleviates neuronal death and axon demyelination after HIBI in neonatal rats by regulating microglia autophagy and cathepsin B expression, and therefore provided long-term cognitive, learning and memory protection.

Disruption of oligodendrocyte progenitor cells is an early sign of pathology in the triple transgenic mouse model of Alzheimer's disease

There is increasing evidence that myelin disruption is related to cognitive decline in Alzheimer's disease (AD). In the CNS, myelin is produced by oligodendrocytes, which are generated throughout life by adult oligodendrocyte progenitor cells (OPCs), also known as NG2-glia. To address whether alterations in myelination are related to age-dependent changes in OPCs, we analyzed NG2 and myelin basic protein (MBP) immunolabelling in the hippocampus of 3×Tg-AD mice at 6 and 24 months of age, compared with non-Tg age-matched controls. There was an age-related decrease in MBP immunostaining and OPC density, together with a decline in the number of OPC sister cells, a measure of OPC replication. Notably, the loss of myelin and OPC sister cells occurred earlier at 6 months in 3xTg-AD, suggesting accelerated aging, although there was not a concomitant decline in OPC numbers at this age, suggesting the observed changes in myelin were not a consequence of replicative exhaustion, but possibly of OPC disruption or senescence. In line with this, a key finding is that compared to age-match controls, OPC displayed marked morphological atrophy at 6 months in 3xTg-AD followed by morphological hypertrophy at 24 months, as deduced from significant changes in total cell surface area, total cell volume, somata volume and branching of main processes. Moreover, we show that hypertrophic OPCs surround and infiltrate amyloid-β (Aβ) plaques, a key pathological hallmark of AD. The results indicate that OPCs undergo complex age-related remodeling in the hippocampus of the 3xTg-AD mouse model. We conclude that OPC disruption is an early pathological sign in AD and is a potential factor in accelerated myelin loss and cognitive decline.

CZ-7, a new derivative of Claulansine F, promotes remyelination induced by cuprizone by enhancing myelin debris clearance

The mechanism of demyelinating diseases is controversial, while demyelination and remyeliantion disorder is the acknowledged etiology and therapeutic target. Untill now, there is no efficient therapy for these diseases. CZ-7, a new derivative of Claulansine F, which has been reported before, were investigated its pro-remyelination effect and its associated mechanism in cuprizone (CPZ)-induced demyelination model. In this study, male C57BL/6 mice were subjected to CPZ (300 mg/kg) through intragastric gavage and were orally administered CZ-7 (20 mg/kg) meanwhile. The results of weight monitoring and behavioral testing showed that CZ-7 can significantly improve behavior dysfunction in the demyelinating mice. Luxol-fast blue (LFB) staining, myelin basic protein (MBP) immunostaining, transmission electron microscopy (TEM) and QPCR results indicated the therapeutic effect of CZ-7 on CPZ mice model. Furthermore, degraded myelin basic protein (dMBP) immunofluorescent staining and oil red O staining showed that CZ-7 contributed to the clearance of degraded myelin debris. More microglia displayed phagocytic shape assembled in corpus callosum (CC) and there was an active process of phagocytosis in microglia after CZ-7 treatment. Immunofluorescent staining and QPCR analysis revealed the M2-polarized phenotype switch of microglia in the process of myelin debris removel, which demostrated the microenvironment improvement of CZ-7. Moreover, immunofluorescent staining of NG2 and O4 demonstated that more oligodendrocyte precursor cells (OPCs) existed in CC after CZ-7 treatment. In conclusion, our results demonstrated CZ-7 has a potential therapeutic effect for MS and other demyelinating diseases through enhancing myelin debris clearance to improve the microenvironment.

Combined Diffusion Tensor Imaging and Quantitative Susceptibility Mapping Discern Discrete Facets of White Matter Pathology Post-injury in the Rodent Brain.

Early loss of white matter microstructure integrity is a significant cause of long-term neurological disorders following traumatic brain injury (TBI). White matter abnormalities typically involve axonal loss and demyelination. In-vivo imaging tools to detect and differentiate such microstructural changes are not well-explored. This work utilizes the conjoint potential offered by advanced magnetic resonance imaging techniques, including quantitative susceptibility mapping (QSM) and diffusion tensor imaging (DTI), to discern the underlying white matter pathology at specific time points (5 h, 1, 3, 7, 14, and 30 days) post-injury in the controlled cortical impact mouse model. A total of 42 animals were randomized into six TBI groups (n = 6 per group) and one sham group (n = 6). Histopathology was performed to validate in-vivo findings by performing myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) immunostaining for the assessment of changes to myelin and astrocytes. After 5 h of injury radial diffusivity (RD) was increased in white matter without a significant change in axial diffusivity (AxD) and susceptibility values. After 1 day post-injury RD was decreased. AxD and susceptibility changes were seen after 3 days post-injury. Susceptibility increases in white matter were observed in both ipsilateral and contralateral regions and persisted for 30 days. In histology, an increase in GFAP immunoreactivity was observed after 3 days post-injury and remained high for 30 days in both ipsilateral and contralateral white matter regions. A loss in MBP signal was noted after 3 days post-injury that continued up to 30 days. In conclusion, these results demonstrate the complementary ability of DTI and QSM in discerning the micro-pathological processes triggered following TBI. While DTI revealed acute and focal white matter changes, QSM mirrored the temporal demyelination in the white matter tracts and diffuse regions at the chronic state.

Longitudinal evaluation of demyelinated lesions in a multiple sclerosis model using ultrashort echo time magnetization transfer (UTE-MT) imaging

Alterations in myelin integrity are involved in many neurological disorders and demyelinating diseases, such as multiple sclerosis (MS). Although magnetic resonance imaging (MRI) is the gold standard method to diagnose and monitor MS patients, clinically available MRI protocols show limited specificity for myelin detection, notably in cerebral grey matter areas. Ultrashort echo time (UTE) MRI has shown great promise for direct imaging of lipids and myelin sheaths, and thus holds potential to improve lesion detection.In this study, we used a sequence combining magnetization transfer (MT) with UTE (“UTE-MT”, TE ​= ​76 ​μs) and with short TE (“STE-MT”, TE ​= ​3000 ​μs) to evaluate spatial and temporal changes in brain myelin content in the cuprizone mouse model for MS on a clinical 7 ​T scanner. During demyelination, UTE-MT ratio (UTE-MTR) and STE-MT ratio (STE-MTR) values were significantly decreased in most white matter and grey matter regions. However, only UTE-MTR detected cortical changes. After remyelination in subcortical and cortical areas, UTE-MTR values remained lower than baseline values, indicating that UTE-MT, but not STE-MT, imaging detected long-lasting changes following a demyelinating event.Next, we evaluated the potential correlations between imaging values and underlying histopathological markers. The strongest correlation was observed between UTE-MTR and percent coverage of myelin basic protein (MBP) immunostaining (r2 ​= ​0.71). A significant, although lower, correlation was observed between STE-MTR and MBP (r2 ​= ​0.48), and no correlation was found between UTE-MTR or STE-MTR and gliosis immunostaining. Interestingly, correlations varied across brain substructures.Altogether, our results demonstrate that UTE-MTR values significantly correlate with myelin content as measured by histopathology, not only in white matter, but also in subcortical and cortical grey matter regions in the cuprizone mouse model for MS. Readily implemented on a clinical 7 ​T system, this approach thus holds great potential for detecting demyelinating/remyelinating events in both white and grey matter areas in humans. When applied to patients with neurological disorders, including MS patient populations, UTE-MT methods may improve the non-invasive longitudinal monitoring of brain lesions, not only during disease progression but also in response to next generation remyelinating therapies.

Phloroglucinol derivative compound 21 attenuates cuprizone-induced multiple sclerosis mice through promoting remyelination and inhibiting neuroinflammation.

Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease in the central nervous system. The myelin loss is mainly caused by dysfunction of oligodendrocytes and inflammatory responses of microglia and astrocytes further aggravate the demyelination. Current therapies for MS focus on suppressing the overactivated immune response but cannot halt the disease progress, so effective drugs are urgently needed. Compound 21 is a phloroglucinol derivative that has been proved to have an outstanding anti-inflammatory effect. The purpose of the present study is to investigate whether this novel compound is effective in MS. The cuprizone-induced model was used in this study to mimic the pathological progress of MS. The results showed that Compound 21 significantly improved the neurological dysfunction and motor coordination impairment. Luxol Fast Blue staining and myelin basic protein immunostaining demonstrated that Compound 21 remarkably promoted remyelination. In addition, Compound 21 significantly promoted oligodendrocytes differentiation. Furthermore, we found that Compound 21 decreased microglia and astrocytes activities and the subsequent neuroinflammatory response, indicating that the anti-inflammatory effect of Compound 21 was also involved in its neuro-protection. All the data prove that Compound 21 exerts protective effect on MS through promoting remyelination and suppressing neuroinflammation, indicating that Compound 21 might be a potential drug candidate for MS treatment.

Glucocorticoid-Induced Reductions of Myelination and Connexin 43 in Mixed Central Nervous System Cell Cultures Are Prevented by Mifepristone

Repeated stress induces systemic elevations in glucocorticoid levels. Stress is also associated with alterations in central nervous system astrocytes and oligodendrocytes that involve connexins and myelin proteins. Corticosteroid elevation seems a major factor in stress-induced neuropathology. Changes in astrocyte connexins and myelin components may be important mediators for the neurological effects of corticosteroid elevations. Two primary cell culture models, myelination culture from rat embryonic spinal cord (SC) or cerebral cortex (CC) consisting of neurons and glial cells (oligodendrocytes, microglia and astrocytes), and mixed astrocyte-and-oligodendrocyte culture prepared from postnatal rat CC, were used in this study. Cell cultures were treated with either vehicle, corticosterone (CORT) with or without glucocorticoid receptor antagonist mifepristone, or dexamethasone (DEX) during the period of in vitro myelination. Immunoreactivity of astrocyte connexin 43 (Cx43) and oligodendrocyte myelin basic protein (MBP), or the myelination index (co-localization of MBP and phosphorylated neurofilament) was determined by double immunofluorescent labeling. Oligodendrocyte morphology was evaluated by Sholl analysis. Prolonged exposure to CORT or DEX induced dose-dependent reduction of the myelination index, and of immunostaining for MBP and Cx43 in SC and CC myelination cultures, which was prevented by mifepristone. In glial cultures single CORT or DEX exposure caused shrinkage and simplification of/' MBP- or CNPase-positive oligodendrocyte processes. The results support that concurrent effects of glucocorticoids on myelination and astrocyte Cx43 immunoreactivity are mediated by glucocorticoid receptors and may partially account for the involvement of CNS glia in the pathological effects of prolonged stress.

Reciprocal nerve staining (RNS) for the concurrent detection of choline acetyltransferase and myelin basic protein on paraffin-embedded sections

BackgroundObjective of our work was to develop a sequential double nonfluorescent immunostaining method which allows the selective identification of myelinated motor fibers in paraffin-embedded samples of peripheral nerves. Motor recovery after a nerve gap-lesion repaired by artificial nerve-guides ("conduits") is often less complete and slower than sensory recovery. The mechanism for this is not fully understood. New methodIncubation in sheep polyclonal choline acetyltransferase antibody (Abcam 18,736) at dilution of 1:150 was followed by incubation in mouse monoclonal anti-myelin basic protein antibody (Abcam 62,631) at a dilution of 1:5000. Counterstaining was performed with hematoxylin QS (Vector Labs H-3404). ResultsImmunostaining of choline acetyltransferase and myelin basic protein can be combined together and results show a good contrast between the light brown of the choline acetyltransferase reaction product and the green of myelin basic protein reaction product. Cell nuclei are stained blue.This new protocol retains the advantages of paraffin embedded sections such as (i) having a relatively simple methodology, (ii) years-long storage life, and (iii) easy sharing among laboratories.Comparison with existing method. This specific combinatorial protocol has never been used before on paraffin embedded sections. It has been named "reciprocal nerve staining" (RNS). ConclusionsRoutine combination of choline acetyltransferase and myelin basic protein immunostaining provides a highly specific, highly contrasted paraffin-embedded sections where optical differentiation of myelinated motor fibers is easy and straightforward. This method will likely simplify and speed-up the routine histological study of nerve regeneration and will contribute a better identification of the nerve motor component.

Neurosteroid replacement therapy using the allopregnanolone-analogue ganaxolone following preterm birth in male guinea pigs.

Children born preterm, especially boys, are at increased risk of developing attention deficit hyperactivity disorder (ADHD) and learning difficulties. We propose that neurosteroid-replacement therapy with ganaxolone (GNX) following preterm birth may mitigate preterm-associated neurodevelopmental impairment. Time-mated sows were delivered preterm (d62) or at term (d69). Male preterm pups were randomized to ganaxolone (Prem-GNX; 2.5 mg/kg subcutaneously twice daily until term equivalence), or preterm control (Prem-CON). Surviving male juvenile pups underwent behavioural testing at d25-corrected postnatal age (CPNA). Brain tissue was collected at CPNA28 and mature myelinating oligodendrocytes of the hippocampus and subcortical white matter were quantified by immunostaining of myelin basic protein (MBP). Ganaxolone treatment returned the hyperactive behavioural phenotype of preterm-born juvenile males to a term-born phenotype. Deficits in MBP immunostaining of the preterm hippocampus and subcortical white matter were also ameliorated in animals receiving ganaxolone. However, during the treatment period weight gain was poor, and pups were sedated, ultimately increasing the neonatal mortality rate. Ganaxolone improved neurobehavioural outcomes in males suggesting that neonatal treatment may be an option for reducing preterm-associated neurodevelopmental impairment. However, dosing studies are required to reduce the burden of unwanted side effects.

Ontogeny of white matter, toll-like receptor expression, and motor skills in the neonatal ferret

Inflammation caused by perinatal infection, superimposed with hypoxia and/or hyperoxia, appears to be important in the pathogenesis of preterm neonatal encephalopathy, with white matter particularly vulnerable during the third trimester. The associated inflammatory response is at least partly mediated through Toll-like receptor (TLR)-dependent mechanisms. Immunohistochemistry, gene expression, and behavioral studies were used to characterize white matter development and determine TLR3 and TLR4 expression and accumulation in the neonatal ferret brain. Expression of markers of white matter development increased significantly between postnatal day (P)1 and P10 (NG2, PDGFRα) or P15 (Olig2), and either remained elevated (NG2), or decreased again at P40 (PDGFRα, Olig2). Olig2 immunostaining within the internal capsule was also greatest at P15. Myelin basic protein (MBP) immunostaining and mRNA expression increased markedly from P15 to P40 and into adulthood, which correlated with increasing performance on behavioral tests (negative geotaxis, cliff aversion, righting reflex, and catwalk gait analysis). TLR4 and TLR3 positive staining was low at all ages, but TLR3 and TLR4 mRNA expression both increased significantly from P1 to P40. Following lipopolysaccharide (LPS) and hypoxia/hyperoxia exposure at P10, meningeal and parenchymal inflammation was seen, including an increase in TLR4 positive cells. These data suggest that the neuroinflammation associated with prematurity could be modeled in the newborn ferret.