The comparative effects of bone marrow mesenchymal stem cells and supernatant transplantation on demyelination and inflammation in cuprizone model.

BACKGROUND: Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) with inflammation and immune dysfunction. OBJECTIVES: We compared the remyelination and immunomodulation properties of mesenchymal stem cells (MSCs) with their conditioned medium (CM) in the cuprizone model. METHODS: Twenty-four C57BL/ 6 mice were divided into four groups. After cuprizone demyelination, MSCs and their CM were injected into the right lateral ventricle of mice. The expression level of IL-1ß, TNF-α, and BDNF genes was evaluated using the qRT-PCR. APC antibody was used to assess the oligodendrocyte population using the immunofluorescent method. The remyelination and axonal repair were studied by specific staining of the LFB and electron microscopy techniques. RESULTS: Transplantation of MSCs and CM increased the expression of the BDNF gene and decreased the expression of IL-1ß and TNF-α genes compared to the cuprizone group, and these effects in the cell group were more than CM. Furthermore, cell transplantation resulted in a significant improvement in myelination and axonal repair, which was measured by luxol fast blue and transmission electron microscope images. The cell group had a higher number of oligodendrocytes than other groups. CONCLUSIONS: According to the findings, injecting MSCs intraventricularly versus cell-conditioned medium can be a more effective approach to improving chronic demyelination in degenerative diseases like MS.

Assessing neuroprotective effects of diroximel fumarate and siponimod via modulation of pacemaker channels in an experimental model of remyelination.

Cuprizone (CPZ)-induced alterations in axonal myelination are associated with a period of neuronal hyperexcitability and increased activity of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in the thalamocortical (TC) system. Substances used for the treatment of multiple sclerosis (MS) have been shown to normalize neuronal excitability in CPZ-treated mice. Therefore, we aimed to examine the effects of diroximel fumarate (DRF) and the sphingosine 1-phospate receptor (S1PR) modulator siponimod on action potential firing and the inward current (Ih) carried by HCN ion channels in naive conditions and during different stages of de- and remyelination. Here, DRF application reduced Ih current density in ex vivo patch clamp recordings from TC neurons of the ventrobasal thalamic complex (VB), thereby counteracting the increase of Ih during early remyelination. Siponimod reduced Ih in VB neurons under control conditions but had no effect in neurons of the auditory cortex (AU). Furthermore, siponimod increased and decreased AP firing properties of neurons in VB and AU, respectively. Computational modeling revealed that both DRF and siponimod influenced thalamic bursting during early remyelination by delaying the onset and decreasing the interburst frequency. Thus, substances used in MS treatment normalize excitability in the TC system by influencing AP firing and Ih.

An Association of Chitinase-3 Like-Protein-1 With Neuronal Deterioration in Multiple Sclerosis.

Elevated levels of Chitinase-3-like protein-1 (CHI3L1) in cerebrospinal fluid have previously been linked to inflammatory activity and disease progression in multiple sclerosis (MS) patients. This study aimed to investigate the presence of CHI3L1 in the brains of MS patients and in the cuprizone model in mice (CPZ), a model of toxic/metabolic demyelination and remyelination in different brain areas. In MS gray matter (GM), CHI3L1 was detected primarily in astrocytes and in a subset of pyramidal neurons. In neurons, CHI3L1 immunopositivity was associated with lipofuscin-like substance accumulation, a sign of cellular aging that can lead to cell death. The density of CHI3L1-positive neurons was found to be significantly higher in normal-appearing MS GM tissue compared to that of control subjects (p = .014). In MS white matter (WM), CHI3L1 was detected in astrocytes located within lesion areas, as well as in perivascular normal-appearing areas and in phagocytic cells from the initial phases of lesion development. In the CPZ model, the density of CHI3L1-positive cells was strongly associated with microglial activation in the WM and choroid plexus inflammation. Compared to controls, CHI3L1 immunopositivity in WM was increased from an early phase of CPZ exposure. In the GM, CHI3L1 immunopositivity increased later in the CPZ exposure phase, particularly in the deep GM region. These results indicate that CHI3L1 is associated with neuronal deterioration, pre-lesion pathology, along with inflammation in MS.

Brain Stiffness Follows Cuprizone-Induced Variations in Local Myelin Content.

Brain maturation and neurological diseases are intricately linked to microstructural changes that inherently affect the brain's mechanical behavior. Animal models are frequently used to explore relative brain stiffness changes as a function of underlying microstructure. Here, we are using the cuprizone mouse model to study indentation-derived stiffness changes resulting from acute and chronic demyelination during a 15-week observation period. We focus on the corpus callosum, cingulum, and cortex which undergo different degrees of de- and remyelination and, therefore, result in region-specific stiffness changes. Mean stiffness of the corpus callosum starts at 1.1 ± 0.3 kPa in untreated mice, then cuprizone treatment causes stiffness to drop to 0.6 ± 0.1 kPa by week 3, temporarily increase to 0.9 ± 0.3 kPa by week 6, and ultimately stabilize around 0.7 ± 0.1 kPa by week 9 for the rest of the observation period. The cingulum starts at 3.2 ± 0.9 kPa, then drops to 1.6 ± 0.4 kPa by week 3, and then gradually stabilizes around 1.4 ± 0.3 kPa by week 9. Cortical stiffness exhibits less stiffness variations overall; it starts at 4.2 ± 1.3 kPa, drops to 2.4 ± 0.6 kPa by week 3, and stabilizes around 2.7 ± 0.9 kPa by week 6. We also assess the impact of tissue fixation on indentation-based mechanical tissue characterization. On the one hand, fixation drastically increases untreated mean tissue stiffness by a factor of 3.3 for the corpus callosum, 2.9 for the cingulum, and 3.6 for the cortex; on the other hand, fixation influences interregional stiffness ratios during demyelination, thus suggesting that fixation affects individual brain tissues differently. Lastly, we determine the spatial correlation between stiffness measurements and myelin density and observe a region-specific proportionality between myelin content and tissue stiffness. STATEMENT OF SIGNIFICANCE: Despite extensive work, the relationship between microstructure and mechanical behavior in the brain remains mostly unknown. Additionally, the existing variation of measurement results reported in literature requires in depth investigation of the impact of individual cell and protein populations on tissue stiffness and interregional stiffness ratios. Here, we used microindentation measurements to show that brain stiffness changes with myelin density in the cuprizone-based demyelination mouse model. Moreover, we explored the impact of tissue fixation prior to mechanical characterization because of conflicting results reported in literature. We observe that fixation has a distinctly different impact on our three regions of interest, thus causing region-specific tissue stiffening and, more importantly, changing interregional stiffness ratios.

A Dual Therapy of Nanostructured Lipid Carrier Loaded with Teriflunomide-A Dihydro-Orotate Dehydrogenase Inhibitor and an miR-155-Antagomir in Cuprizone-Induced C57BL/6J Mouse.

The effective treatment of central nervous system (CNS) disorders such as multiple sclerosis (MS) has been challenging due to the limited ability of therapeutic agents to cross the blood-brain barrier (BBB). In this study, we investigated the potential of nanocarrier systems to deliver miR-155-antagomir-teriflunomide (TEF) dual therapy to the brain via intranasal (IN) administration to manage MS-associated neurodegeneration and demyelination. Our results showed that the combinatorial therapy of miR-155-antagomir and TEF loaded in nanostructured lipid carriers (NLCs) significantly increased brain concentration and improved targeting potential. The novelty of this study lies in the use of a combinatorial therapy approach of miR-155-antagomir and TEF loaded in NLCs. This is a significant finding, as the effective delivery of therapeutic molecules to the CNS has been a challenge in treating neurodegenerative disorders. Additionally, this study sheds light on the potential use of RNA-targeting therapies in personalized medicine, which could revolutionize the way CNS disorders are managed. Furthermore, our findings suggest that nanocarrier-loaded therapeutic agents have great potential for safe and economical delivery in treating CNS disorders. Our study provides novel insights into the effective delivery of therapeutic molecules via the IN route for managing neurodegenerative disorders. In particular, our results demonstrate the potential of delivering miRNA and TEF via the intranasal route using the NLC system. We also demonstrate that the long-term use of RNA-targeting therapies could be a promising tool in personalized medicine. Importantly, using a cuprizone-induced animal model, our study also investigated the effects of TEF-miR155-antagomir-loaded NLCs on demyelination and axonal damage. Following six weeks of treatment, the TEF-miR155-antagomir-loaded NLCs potentially lowered the demyelination and enhanced the bioavailability of the loaded therapeutic molecules. Our study is a paradigm shift in delivering miRNAs and TEF via the intranasal route and highlights the potential of this approach for managing neurodegenerative disorders. In conclusion, our study provides critical insights into the effective delivery of therapeutic molecules via the IN route for managing CNS disorders, and especially MS. Our findings have significant implications for the future development of nanocarrier-based therapies and personalized medicine. Our results provide a strong foundation for further studies and the potential to develop safe and economic therapeutics for CNS disorders.

Huperzine-A Improved Animal Behavior in Cuprizone-Induced Mouse Model by Alleviating Demyelination and Neuroinflammation.

Huperzine A (HupA) is a natural acetylcholinesterase inhibitor (AChEI) with the advantages of high efficiency, selectivity as well as reversibility and can exhibit significant therapeutic effects against certain neurodegenerative diseases. It is also beneficial in reducing the neurological impairment and neuroinflammation of experimental autoimmune encephalomyelitis (EAE), a classic model for multiple sclerosis (MS). However, whether HupA can directly regulate oligodendrocyte differentiation and maturation and promote remyelination has not been investigated previously. In this study, we have analyzed the potential protective effects of HupA on the demylination model of MS induced by cuprizone (CPZ). It was found that HupA significantly attenuated anxiety-like behavior, as well as augmented motor and cognitive functions in CPZ mice. It also decreased demyelination and axonal injury in CPZ mice. Moreover, in CPZ mice, HupA increased mRNA levels of the various anti-inflammatory cytokines (Arg1, CD206) while reducing the levels of different pro-inflammatory cytokines (iNOS, IL-1ß, IL-18, CD16, and TNF-α). Mecamylamine, a nicotinic acetylcholinergic receptor antagonist, could effectively reverse the effects of HupA. Therefore, we concluded that HupA primarily exerts its therapeutic effects on multiple sclerosis through alleviating demyelination and neuroinflammation.

Effects of Axonal Demyelination, Inflammatory Cytokines and Divalent Cation Chelators on Thalamic HCN Channels and Oscillatory Bursting.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1ß; IL-6; INF-α; INF-ß) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (Ih) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of Ih (potential of half-maximal activation (V0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, Ih was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-α and IL-1ß. It was found that Ih parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1ß increased the availability of HCN channels (depolarized V0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-α mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1ß and INF-α increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines.

Different Methods for Evaluating Microglial Activation Using Anti-Ionized Calcium-Binding Adaptor Protein-1 Immunohistochemistry in the Cuprizone Model.

Microglia play an important role in the pathology of various central nervous system disorders, including multiple sclerosis (MS). While different methods exist to evaluate the extent of microglia activation, comparative studies investigating the sensitivity of these methods are missing for most models. In this study, we systematically evaluated which of the three commonly used histological methods (id est, quantification of microglia density, densitometrically evaluated staining intensity, or cellular morphology based on the determination of a ramification index, all measured in anti-ionized calcium-binding adaptor protein-1 (IBA1) immunohistochemical stains) is the most sensitive method to detect subtle changes in the microglia activation status in the context of MS. To this end, we used the toxin-induced cuprizone model which allows the experimental induction of a highly reproducible demyelination in several central nervous system regions, paralleled by early microglia activation. In this study, we showed that after 3 weeks of cuprizone intoxication, all methods reveal a significant microglia activation in the white matter corpus callosum. In contrast, in the affected neocortical grey matter, the evaluation of anti-IBA1 cell morphologies was the most sensitive method to detect subtle changes of microglial activation. The results of this study provide a useful guide for future immunohistochemical evaluations in the cuprizone and other neurodegenerative models.

Visual Evoked Potentials to Monitor Myelin Cuprizone-Induced Functional Changes.

The visual system is one of the most accessible routes to study the central nervous system under pathological conditions, such as in multiple sclerosis (MS). Non-invasive visual evoked potential (VEP) and optical coherence tomography (OCT) were used to assess visual function and neuroretinal thickness in C57BL/6 taking 0.2% cuprizone for 7 weeks and at 5, 8, 12, and 15 days after returning to a normal diet. VEPs were significantly delayed starting from 4 weeks on cuprizone, with progressive recovery off cuprizone, becoming significant at day 8, complete at day 15. In contrast, OCT and neurofilament staining showed no significant axonal thinning. Optic nerve histology indicated that whilst there was significant myelin loss at 7 weeks on the cuprizone diet compared with healthy mice, at 15 days off cuprizone diet demyelination was significantly less severe. The number of Iba 1+ cells was found increased in cuprizone mice at 7 weeks on and 15 days off cuprizone. The combined use of VEPs and OCT allowed us to characterize non-invasively, in vivo, the functional and structural changes associated with demyelination and remyelination in a preclinical model of MS. This approach contributes to the non-invasive study of possible effective treatments to promote remyelination in demyelinating pathologies.

The effects of mesenchymal stem cells transplantation on A1 neurotoxic reactive astrocyte and demyelination in the cuprizone model.

Multiple sclerosis (MS), which is an autoimmune disease, is characterized by symptoms such as demyelination, axonal damage, and astrogliosis. As the most abundant type of glial cells, astrocytes play an important role in MS pathogenesis. Mesenchymal stem cells (MSCs) are a subset of stromal cells that have the potential for migration, immune-modulation, differentiation, remyelination, and neuroregeneration. Therefore, the present study evaluates the effects of MSC transplantation on A1 reactive astrocytes and the remyelination process in the cuprizone mouse model. The study used 30 male C57BL/6 mice, which were randomly distributed into three subgroups (n = 10), i.e., control, cuprizone, and transplanted MSCs groups. In order to generate a chronic demyelination model, the mice in the cuprizone group received food mixed with 0.2% cuprizone powder for 12 weeks. Then, 2 µl of DMEM containing approximately 3 × 105 DiI labeled cells was injected with a 4-min interval into the right lateral ventricle using a 10-µl Hamilton syringe. After 2 weeks of cell transplantation, we used the rotarod test to evaluate the behavioral deficits, while the remyelination process was assessed by transmission electron microscopy (TEM) and Luxol Fast Blue (LFB) staining. We assessed the population of A1 astrocytes and oligodendrocytes using specific markers, such as C3, GFAP, and Olig2, using the immunefleurocent method. The pro-inflammatory and trophic factors were assessed by a real-time polymerase chain reaction. According to our data, the specific marker of A1 astrocytes (C3) decreased in the MSCs group, while the number of oligodendrocytes significantly increased in this group compared to the cuprizone mice. Additionally, MSC was able to enhance the remyelination process after cuprizone usage, as shown by LFB and TEM images. The molecular results showed that MSCs could reduce pro-inflammatory factors, such as IL-1 and TNF-α, through the secretion of BDNF and TGF-ß as trophic factors. The obtained results indicated that MSC could reduce demyelination and inflammation by decreasing A1 neurotoxic reactive astrocytes and neurotrophic and immunomodulatory factors secretion in the chronic cuprizone demyelination model.

EBI2 is expressed in glial cells in multiple sclerosis lesions, and its knock-out modulates remyelination in the cuprizone model.

EBI2 receptor regulates the immune system, and in multiple, sclerosis is upregulated in the central nervous system infiltrating lymphocytes. In newborn EBI2-deficient mice, myelin development is delayed, and its persistent antagonism inhibits remyelination in chemically demyelinated organotypic cerebellar slices. We used the cuprizone model of multiple sclerosis to elucidate the role of central nervous system-expressed EBI2 in de- and remyelination. The wild-type and EBI2 knock-out mice were fed 0.2% cuprizone in chow for 5 weeks and allowed to recover on a normal diet for 2 weeks. The data showed less efficient recovery of myelin, attenuated oligodendrocyte loss, fewer astrocytes and increased total cholesterol levels in the EBI2 knock-out mice after recovery. Moreover, the wild-type mice upregulated EBI2 expression after recovery confirming the involvement of EBI2 signalling during recovery from demyelination in the cuprizone model. The pro-inflammatory cytokine levels were at comparable levels in the wild-type and EBI2 knock-out mice, with only minor differences in TNFα and IL1ß levels either at peak or during recovery. The neuroinflammatory signalling molecules, Abl1 kinase and NFКB1 (p105/p50) subunit, were significantly downregulated in the EBI2 knock-out mice at peak of disease. Immunohistochemical investigations of EBI2 receptor distribution in the central nervous system (CNS) cells in multiple sclerosis (MS) brain revealed strong expression of EBI2 in astrocytes and microglia inside the plaques implicating glia-expressed EBI2 in multiple sclerosis pathophysiology. Taken together, these findings demonstrate the involvement of EBI2 signalling in the recovery from demyelination rather than in demyelination and as such warrant further research into the role of EBI2 in remyelination.

Gut Microbiota Changes in Experimental Autoimmune Encephalomyelitis and Cuprizone Mice Models.

Multiple sclerosis (MS) is a chronic and neurodegenerative disease of the central nervous system (CNS) characterized by the immune mediated attack on axons and the subsequent demyelination. There is growing evidence that the gut microbiota of MS patients is altered; however, the connection between demyelination events and changes in the gut microbiota has not been determined. The objective of the current work was to characterize the microbial dysbiosis in two murine demyelinating models and to study the correlation between them. Concurrently, their suitability as predictors of microbial changes in MS patients was assessed. To this purpose, experimental autoimmune encephalomyelitis (EAE) and cuprizone (CPZ) models were induced in C57BL/6 mice that were monitored for 4 and 9 weeks, respectively. Fecal samples were collected during disease progression. Motor skill performance was evaluated by EAE scale measurement in EAE mice and demyelination by magnetic resonance imaging (MRI) in CPZ ones. EAE and CPZ mice revealed drastic microbial changes according to disease progression, adding a new layer of complexity to the understanding of demyelination and remyelination processes. Besides, the reported microbial changes replicate most of the characteristics that define the potential dysbiosis in MS patients. The controlled environment and stable diet that animals have in research centers offer an exceptional scenario to modify animal's microbiota and provide opportunities to study host microbiota interplay with restrained conditions not achievable in human studies. Nevertheless the slight differences from murine model's and patient's microbiota should be considered in the design of studies aiming to modulate the microbiota.

Inflammation-driven glial alterations in the cuprizone mouse model probed with diffusion-weighted magnetic resonance spectroscopy at 11.7 T.

Inflammation of brain tissue is a complex response of the immune system to the presence of toxic compounds or to cell injury, leading to a cascade of pathological processes that include glial cell activation. Noninvasive MRI markers of glial reactivity would be very useful for in vivo detection and monitoring of inflammation processes in the brain, as well as for evaluating the efficacy of personalized treatments. Due to their specific location in glial cells, myo-inositol (mIns) and choline compounds (tCho) seem to be the best candidates for probing glial-specific intra-cellular compartments. However, their concentrations quantified using conventional proton MRS are not specific for inflammation. In contrast, it has been recently suggested that mIns intra-cellular diffusion, measured using diffusion-weighted MRS (DW-MRS) in a mouse model of reactive astrocytes, could be a specific marker of astrocytic hypertrophy. In order to evaluate the specificity of both mIns and tCho diffusion to inflammation-driven glial alterations, we performed DW-MRS in a volume of interest containing the corpus callosum and surrounding tissue of cuprizone-fed mice after 6 weeks of intoxication, and evaluated the extent of astrocytic and microglial alterations using immunohistochemistry. Both mIns and tCho apparent diffusion coefficients were significantly elevated in cuprizone-fed mice compared with control mice, and histologic evaluation confirmed the presence of severe inflammation. Additionally, mIns and tCho diffusion showed, respectively, strong and moderate correlations with histological measures of astrocytic and microglial area fractions, confirming DW-MRS as a promising tool for specific detection of glial changes under pathological conditions.

The Cuprizone Model: Dos and Do Nots.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. Various pre-clinical models with different specific features of the disease are available to study MS pathogenesis and to develop new therapeutic options. During the last decade, the model of toxic demyelination induced by cuprizone has become more and more popular, and it has contributed substantially to our understanding of distinct yet important aspects of the MS pathology. Here, we aim to provide a practical guide on how to use the cuprizone model and which pitfalls should be avoided.

Nrf2 deficiency increases oligodendrocyte loss, demyelination, neuroinflammation and axonal damage in an MS animal model.

Oxidative stress is a pathophysiological hallmark of many CNS diseases, among multiple sclerosis (MS). Accordingly, boosting the astrocytic transcription factor nuclear factor E2-related factor 2 (Nrf2) system in an MS mouse model efficiently ameliorates oligodendrocyte loss, neuroinflammation and axonal damage. Moreover, Dimethylfumarate, an efficient activator of Nrf2, has recently been approved as therapeutic option in MS treatment. Here, we use the cuprizone mouse model of MS to induce oxidative stress, selective oligodendrocyte loss, microglia and astrocyte activation as well as axonal damage in both wild type and Nrf2-deficient mice. We found increased oligodendrocyte apoptosis and loss, pronounced neuroinflammation and higher levels of axonal damage in cuprizone-fed Nrf2-deficient animals when compared to wild type controls. In addition, Nrf2-deficient animals showed a higher susceptibility towards cuprizone within the commissura anterior white matter tract, a structure that is relatively insensitive to cuprizone in wild type animals. Our data highlight the cuprizone model as a suitable tool to study the complex interplay of oxidative stress, neuroinflammation and axonal damage. Further studies will have to show whether distinct expression patterns of Nrf2 are involved in the variable susceptibility towards cuprizone in the mouse.

Imaging and Quantification of Myelin Integrity After Injury With Spectral Confocal Reflectance Microscopy.

Developing a high-throughput approach to quantify the extent of myelin integrity in preclinical models of demyelinating diseases will enhance our capacity to identify novel therapies for myelin repair. In light of the technical limitations of electron microscopy and immunohistochemical analyses of myelination, we have utilized a novel imaging technique, spectral confocal reflectance (SCoRe) microscopy. SCoRe takes advantage of the optically reflective properties of compact myelin, allowing the integrity of compact myelin to be quantified over the course of the cuprizone-induced model of central demyelination. We applied SCoRe imaging on fixed frozen brain sections. SCoRe analysis of control mice identified an increase in corpus callosum myelination during the period of cuprizone administration and recovery, suggesting that the normal developmental processes of myelination are ongoing at this time. Importantly, analysis of mice subjected to cuprizone identified a significant reduction in compact myelin in both rostral and caudal corpus callosum compared to age-matched control mice. SCoRe microscopy also allowed the visualization and quantification of the amount of myelin debris in demyelinating lesions. Combining SCoRe imaging with immunohistochemistry, we quantified the amount of myelin debris within IBA-1+ microglia and found that 11% of myelin debris colocalized in microglia irrespective of the callosal regions, with the vast majority of debris outside of microglia. In summary, we have demonstrated that SCoRe microscopy is an effective and powerful tool to perform both quantitative and qualitative analyses of compact myelin integrity in health or after injury in vivo, demonstrating its future application in high-throughput assessments and screening of the therapeutic efficacy of myelin repair therapies in preclinical animal models of demyelinating diseases.

Quantitative Imaging of White and Gray Matter Remyelination in the Cuprizone Demyelination Model Using the Macromolecular Proton Fraction.

Macromolecular proton fraction (MPF) has been established as a quantitative clinically-targeted MRI myelin biomarker based on recent demyelination studies. This study aimed to assess the capability of MPF to quantify remyelination using the murine cuprizone-induced reversible demyelination model. MPF was measured in vivo using the fast single-point method in three animal groups (control, cuprizone-induced demyelination, and remyelination after cuprizone withdrawal) and compared to quantitative immunohistochemistry for myelin basic protein (MBP), myelinating oligodendrocytes (CNP-positive cells), and oligodendrocyte precursor cells (OPC, NG2-positive cells) in the corpus callosum, caudate putamen, hippocampus, and cortex. In the demyelination group, MPF, MBP-stained area, and oligodendrocyte count were significantly reduced, while OPC count was significantly increased as compared to both control and remyelination groups in all anatomic structures (p < 0.05). All variables were similar in the control and remyelination groups. MPF and MBP-stained area strongly correlated in each anatomic structure (Pearson's correlation coefficients, r = 0.80-0.90, p < 0.001). MPF and MBP correlated positively with oligodendrocyte count (r = 0.70-0.84, p < 0.01 for MPF; r = 0.81-0.92, p < 0.001 for MBP) and negatively with OPC count (r = -0.69--0.77, p < 0.01 for MPF; r = -0.72--0.89, p < 0.01 for MBP). This study provides immunohistological validation of fast MPF mapping as a non-invasive tool for quantitative assessment of de- and remyelination in white and gray matter and indicates the feasibility of using MPF as a surrogate marker of reparative processes in demyelinating diseases.

Ginkgolide K supports remyelination via induction of astrocytic IGF/PI3K/Nrf2 axis.

Although several therapies are approved, none promote re-myelination in multiple sclerosis (MS) patients, limiting their ability for sustained recovery. Thus, treatment development in MS has the opportunity to tackle the challenges, including experimental therapies targeting neuroprotection and re-myelination. Here, we provide a novel therapeutic target for Ginkgolide K (GK) that is now becoming a very critical natural compound to treat demyelination and neurodegeneration. GK improves behavioral dysfunction and demyelination in cuprizone (CPZ) model, followed by the migration and enrichment of astrocytes in the corpus callosum. Both in vitro and in vivo experiments demonstrates that GK triggers the upregulation of Nrf2/HO-1 in astrocytes and inhibition of p-NF-kB/p65, which is associated with the outcome of anti-inflammation and anti-oxidation by suppressing the production of IL-6 and TNFα as well as nitric oxide and iNOS in astrocytes. Further findings suggest that IGF/PI3K, but not BDNF, was induced in the corpus callosum after GK treatment, revealing that Nrf2 activation inhibited caspase-3 and apoptosis in O4+ oligodendrocytes possibly through IGF/PI3K signaling molecules. Since the current immunomodulatory therapies for MS have failed to prevent patients from entering the progressive phase of the disease, thus targeting Nrf2 in astrocytes with GK would be an ideal strategy for myelin protection and regeneration.

Copper chelation and autoimmunity differentially impact myelin in the hippocampal-prefrontal circuit.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. About 50% of MS patients develop deficits in learning, memory and executive function, which are accompanied by demyelinating lesions in the hippocampus and/or prefrontal cortex (PFC). Why demyelination in these regions occurs in some patients but not in others and what is the underlying mechanism remain unclear. Here we report that myelin density in the hippocampus and PFC is markedly reduced in the cuprizone model, but not in the chronic experimental autoimmune encephalomyelitis. These two models can be used for studying different neuropathophysiological aspects of demyelinating diseases.

Mesenchymal stem cell mediated effects on microglial phenotype in cuprizone-induced demyelination model.

Microglial cells have an essential role in neurodegenerative disorders, such as multiple sclerosis. They are divided into two subgroups: M1 and M2 phenotypes. Mesenchymal stem cells (MSC), with neuroprotective and immunomodulating properties, could improve these diseases. We evaluate the immunomodulating effects of MSC on microglial phenotypes and the improvement of demyelination in a cuprizone (CPZ) model of multiple sclerosis (MS). For inducing the chronic demyelination model, C57BL6 mice were given a diet with 0.2% CPZ (w/w) for 12 weeks. In the MSC group, cells were transplanted into the right lateral ventricle of mice. The expression of targeted genes was assessed by real-time polymerase chain reaction. M1 and M2 microglial phenotypes were assessed by immunohistochemistry of inducible nitric oxide synthase (iNOS) and Arg-1, respectively. Remyelination was studied by luxal fast blue (LFB) staining and electron microscopy (EM). We found that MSC transplantation reduced the expression level of M1-specific messenger RNA (mRNA; iNOS and CD86) but increased the expression level of M2 specific genes (CD206, Arg-1, and CX3CR1) in comparison to the CPZ group. Moreover, cell therapy significantly decreased the M1 marker (iNOS+ cells), but M2 marker (Arg-1+ cells) significantly increased in comparison with the CPZ group. In addition, MSC treatment significantly increased the CX3CL1 expression level in comparison with the CPZ group and led to improvement in remyelination, which was confirmed by LFB and EM images. The results showed that MSC transplantation increases the M2 and decreases the M1 phenotype in MS. This change was accompanied by decrease in demyelination and axonal injury and indicated that MSCs have a positive effect on MS by modification of microglia cells.