The Effects of Cuprizone on Murine Subventricular Zone-Derived Neural Stem Cells and Progenitor Cells Grown as Neurospheres.

Despite the extensive use of the cuprizone (CPZ) demyelination animal model, there is little evidence regarding the effects of CPZ on a cellular level. Initial studies have suggested that oligodendrocytes (OL) are the main cell targets for CPZ toxicity. However, recent data have revealed additional effects on neural stem cells and progenitor cells (NSC/NPC), which constitute a reservoir for OL regeneration during brain remyelination. We cultured NSC/NPC as neurospheres to investigate CPZ effects on cell mechanisms which are thought to be involved in demyelination and remyelination processes in vivo. Proliferating NSC/NPC cultures exposed to CPZ showed overproduction of intracellular reactive oxygen species and increased progenitor migration at the expense of a significant inhibition of cell proliferation. Although NSC/NPC survival was not affected by CPZ in proliferative conditions, we found that CPZ-treated cultures undergoing cell differentiation were more prone to cell death than controls. The commitment and cell differentiation towards neural lineages did not seem to be affected by CPZ, as shown by the conserved proportions of OL, astrocytes, and neurons. Nevertheless, when CPZ treatment was performed after cell differentiation, we detected a significant reduction in the number and the morphological complexity of OL, astrogliosis, and neuronal damage. We conclude that, in addition to damaging mature OL, CPZ also reduces NSC/NPC proliferation and activates progenitor migration. These results shed light on CPZ direct effects on NSC proliferation and the progression of in vitro differentiation.

Colony-stimulating factor-1 receptor inhibition attenuates microgliosis and myelin loss but exacerbates neurodegeneration in the chronic cuprizone model.

Multiple sclerosis (MS), especially in its progressive phase, involves early axonal and neuronal damage resulting from a combination of inflammatory mediators, demyelination, and loss of trophic support. During progressive disease stages, a microenvironment is created within the central nervous system (CNS) favoring the arrival and retention of inflammatory cells. Active demyelination and neurodegeneration have also been linked to microglia (MG) and astrocyte (AST)-activation in early lesions. While reactive MG can damage tissue, exacerbate deleterious effects, and contribute to neurodegeneration, it should be noted that activated MG possess neuroprotective functions as well, including debris phagocytosis and growth factor secretion. The progressive form of MS can be modeled by the prolonged administration to cuprizone (CPZ) in adult mice, as CPZ induces highly reproducible demyelination of different brain regions through oligodendrocyte (OLG) apoptosis, accompanied by MG and AST activation and axonal damage. Therefore, our goal was to evaluate the effects of a reduction in microglial activation through orally administered brain-penetrant colony-stimulating factor-1 receptor (CSF-1R) inhibitor BLZ945 (BLZ) on neurodegeneration and its correlation with demyelination, astroglial activation, and behavior in a chronic CPZ-induced demyelination model. Our results show that BLZ treatment successfully reduced the microglial population and myelin loss. However, no correlation was found between myelin preservation and neurodegeneration, as axonal degeneration was more prominent upon BLZ treatment. Concomitantly, BLZ failed to significantly offset CPZ-induced astroglial activation and behavioral alterations. These results should be taken into account when proposing the modulation of microglial activation in the design of therapies relevant for demyelinating diseases. Cover Image for this issue: https://doi.org/10.1111/jnc.15394.

Galectin-3 Exerts a Pro-differentiating and Pro-myelinating Effect Within a Temporal Window Spanning Precursors and Pre-oligodendrocytes: Insights into the Mechanisms of Action.

Oligodendrocytes (OLG) are the cells resident in the CNS responsible for myelination. OLG undergo a succession of morphological and molecular changes along several maturational stages. Galectin-3 (Gal-3) is a 25- to 35-KDa protein belonging to the family of carbohydrate-binding galectins, which bind to glycoconjugates containing ß-galactosides. Gal-3 lacks a specific receptor and its binding is thus rather unspecific, as it depends on the cellular environment and the repertoire of glycomolecules at the time when Gal-3 is present. Our previous work revealed that recombinant Gal-3 (rGal-3)-treated OLG showed accelerated differentiation, evidenced by an increase in the number of mature cells to the detriment of immature ones and accelerated actin cytoskeleton dynamics. These changes were a consequence of rGal-3 influence on Akt, Erk 1/2, and ß-catenin signaling pathways. Considering this previous evidence, the aim of this study was to identify the temporal window of rGal-3 action on the OLG lineage to induce OLG maturation by using specific single pulses of rGal-3 over the different maturational stages of OLG, and to unravel its main direct targets promoting OLG differentiation by mass spectrometry analysis. Our results reveal a key temporal window spanning between OPC and pre-OLG states in which rGal-3 action promotes OLG differentiation, and identify several targets for rGal-3 binding including proteins related to the cytoskeleton, signaling pathways, metabolism and intracellular trafficking, among others. These results highlight the relevance of Gal-3 in signaling pathways regulating oligodendroglial differentiation and support a potential therapeutic role for rGal-3 in demyelinating diseases such as multiple sclerosis.

Five Decades of Cuprizone, an Updated Model to Replicate Demyelinating Diseases.

INTRODUCTION: Demyelinating diseases of the central nervous system (CNS) comprise a group of neurological disorders characterized by progressive (and eventually irreversible) loss of oligodendrocytes and myelin sheaths in the white matter tracts. Some of myelin disorders include: Multiple sclerosis, Guillain-Barré syndrome, peripheral nerve polyneuropathy and others. To date, the etiology of these disorders is not well known and no effective treatments are currently available against them. Therefore, further research is needed to gain a better understand and treat these patients. To accomplish this goal, it is necessary to have appropriate animal models that closely resemble the pathophysiology and clinical signs of these diseases. Herein, we describe the model of toxic demyelination induced by cuprizone (CPZ), a copper chelator that reduces the cytochrome and monoamine oxidase activity into the brain, produces mitochondrial stress and triggers the local immune response. These biochemical and cellular responses ultimately result in selective loss of oligodendrocytes and microglia accumulation, which conveys to extensive areas of demyelination and gliosis in corpus callosum, superior cerebellar peduncles and cerebral cortex. Remarkably, some aspects of the histological pattern induced by CPZ are similar to those found in multiple sclerosis. CPZ exposure provokes behavioral changes, impairs motor skills and affects mood as that observed in several demyelinating diseases. Upon CPZ removal, the pathological and histological changes gradually revert. Therefore, some authors have postulated that the CPZ model allows to partially mimic the disease relapses observed in some demyelinating diseases. CONCLUSION: for five decades, the model of CPZ-induced demyelination is a good experimental approach to study demyelinating diseases that has maintained its validity, and is a suitable pharmacological model for reproducing some key features of demyelinating diseases, including multiple sclerosis.

Microglial modulation through colony-stimulating factor-1 receptor inhibition attenuates demyelination.

Multiple sclerosis (MS) is one of the most common causes of progressive disability affecting young people with very few therapeutic options available for its progressive forms. Its pathophysiology involves demyelination and neurodegeneration apparently driven by microglial activation, which is physiologically dependent on colony-stimulating factor-1 receptor (CSF-1R) signaling. In the present work, we used microglial modulation through oral administration of brain-penetrant CSF-1R inhibitor BLZ945 in acute and chronic cuprizone (CPZ)-induced demyelination to evaluate preventive and therapeutic effects on de/remyelination and neurodegeneration. Our results show that BLZ945 induced a significant reduction in the number of microglia. Preventive BLZ945 treatment attenuated demyelination in the acute CPZ model, mainly in cortex and external capsule. In contrast, BLZ945 treatment in the acute CPZ model failed to protect myelin or foster remyelination in myelin-rich areas, which may respond to a loss in microglial phagocytic capacity and the consequent impairment in oligodendroglial differentiation. Preventive and therapeutic BLZ945 treatment promoted remyelination and neuroprotection in the chronic model. These results could be potentially transferred to the treatment of progressive forms of MS.

Progesterone effects on oligodendrocyte differentiation in injured spinal cord.

Spinal cord lesions result in chronic demyelination as a consequence of secondary injury. Although oligodendrocyte precursor cells proliferate the differentiation program fails. Successful differentiation implies progressive decrease of transcriptional inhibitors followed by upregulation of activators. Progesterone emerges as an anti-inflammatory and pro-myelinating agent which improves locomotor outcome after spinal cord injury. In this study, we have demonstrated that spinal cord injury enhanced oligodendrocyte precursor cell number and decreased mRNA expression of transcriptional inhibitors (Id2, Id4, hes5). However, mRNA expression of transcriptional activators (Olig2, Nkx2.2, Sox10 and Mash1) was down-regulated 3 days post injury. Interestingly, a differentiation factor such as progesterone increased transcriptional activator mRNA levels and the density of Olig2- expressing oligodendrocyte precursor cells. The differentiation program is regulated by extracellular signals which modify transcriptional factors and epigenetic players. As TGFß1 is a known oligodendrocyte differentiation factor which is regulated by progesterone in reproductive tissues, we assessed whether TGFß1 could mediate progesterone remyelinating actions after the lesion. Notwithstanding that astrocyte, oligodendrocyte precursor and microglial cell density increased after spinal cord injury, the number of these cells which expressed TGFß1 remained unchanged regarding sham operated rats. However, progesterone treatment increased TGFß1 mRNA expression and the number of astrocytes and microglial TGFß1 expressing cells which would indirectly enhance oligodendrocyte differentiation. Therefore, TGFß1 arises as a potential mediator of progesterone differentiating effects on oligodendrocyte linage.

Propentofylline reduces glial scar development following gliotoxic damage in the rat brainstem.

OBJECTIVE: This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. METHOD: Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. RESULTS AND CONCLUSION: Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.

Propentofylline reduces glial scar development following gliotoxic damage in the rat brainstem / Propentofilina reduz o desenvolvimento da cicatriz glial após dano gliotóxico no tronco encefálico de ratos

ABSTRACT Propentofylline is a xanthine derivative that depresses activation of glial cells, whose responses contribute to neural tissue damage during inflammation. Ethidium bromide injection into the central nervous system induces local oligodendroglial and astrocytic loss, resulting in primary demyelination, neuroinflammation and blood-brain barrier disruption. Surviving astrocytes present a vigorous reaction around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). Objective This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. Method Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. Results and Conclusion Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.

RESUMO A propentofilina é uma xantina que deprime a ativação das células gliais, cujas respostas contribuem para o dano neural durante inflamação. A injeção de brometo de etídio no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização, neuroinflamação e ruptura da barreira hematoencefálica. Os astrócitos sobreviventes apresentam vigorosa reação ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). Objetivo Este estudo objetivou avaliar o efeito da propentofilina sobre a resposta astrocitária após injúria gliotóxica. Método Ratos Wistar foram injetados com brometo de etídio na cisterna basal e tratados ou não com propentofilina (12.5mg/kg/dia, intraperitoneal). Amostras do tronco encefálico foram coletadas dos 15 aos 31 dias pós-injeção do gliotóxico e processadas para estudo ultraestrutural e imuno-histoquímico para GFAP. Resultados e Conclusão Os resultados demonstram que a propentofilina reduziu a ativação astrocitária até o 21o dia, sugerindo que essa droga pode atuar na redução da cicatriz glial após injúria.

Propentofylline treatment on open field behavior in rats with focal ethidium bromide-induced demyelination in the ventral surface of the brainstem.

Propentofylline (PPF) is a xanthine derivative with pharmacological effects that are distinct from those of classic methylxanthines. It depresses the activation of microglial cells and astrocytes, which is associated with neuronal damage during neural inflammation and hypoxia. Our previous studies showed that PPF improved remyelination following gliotoxic lesions that were induced by ethidium bromide (EB). In the present study, the long-term effects of PPF on open field behavior in rats with EB-induced focal demyelination were examined. The effects of PPF were first evaluated in naive rats that were not subjected to EB lesions. Behavior in the beam walking test was also evaluated during chronic PPF treatment because impairments in motor coordination can interfere with behavior in the open field. The results showed that PPF treatment in unlesioned rats decreased general activity and caused motor impairment in the beam walking test. Gliotoxic EB injections increased general activity in rats that were treated with PPF compared with rats that received saline solution. Motor incoordination was also attenuated in PPF-treated rats. These results indicate that PPF reversed the effects of EB lesions on behavior in the open field and beam walking test.

Paving the way for adequate myelination: The contribution of galectin-3, transferrin and iron.

Considering the worldwide incidence of well characterized demyelinating disorders such as Multiple Sclerosis (MS) and the increasing number of pathologies recently found to involve hypomyelinating factors such as micronutrient deficits, elucidating the molecular basis of central nervous system (CNS) demyelination, remyelination and hypomyelination becomes essential to the development of future neuroregenerative therapies. In this context, this review discusses novel findings on the contribution of galectin-3 (Gal-3), transferrin (Tf) and iron to the processes of myelination and remyelination and their potentially positive regulation of oligodendroglial precursor cell (OPC) differentiation. Studies were conducted in cuprizone (CPZ)-induced demyelination and iron deficiency (ID)-induced hypomyelination, and the participation of glial and neural stem cells (NSC) in the remyelination process was evaluated by means of both in vivo and in vitro assays on primary cell cultures.

Astrocyte Activation via Stat3 Signaling Determines the Balance of Oligodendrocyte versus Schwann Cell Remyelination.

Remyelination within the central nervous system (CNS) most often is the result of oligodendrocyte progenitor cells differentiating into myelin-forming oligodendrocytes. In some cases, however, Schwann cells, the peripheral nervous system myelinating glia, are found remyelinating demyelinated regions of the CNS. The reason for this peripheral type of remyelination in the CNS and what governs it is unknown. Here, we used a conditional astrocytic phosphorylated signal transducer and activator of transcription 3 knockout mouse model to investigate the effect of abrogating astrocyte activation on remyelination after lysolecithin-induced demyelination of spinal cord white matter. We show that oligodendrocyte-mediated remyelination decreases and Schwann cell remyelination increases in lesioned knockout mice in comparison with lesioned controls. Our study shows that astrocyte activation plays a crucial role in the balance between Schwann cell and oligodendrocyte remyelination in the CNS, and provides further insight into remyelination of CNS axons by Schwann cells.

The subventricular zone is able to respond to a demyelinating lesion after localized radiation.

Radiation is a common tool in the treatment of brain tumors that induces neurological deficits as a side effect. Some of these deficits appear to be related to the impact of radiation on the neurogenic niches, producing a drastic decrease in the proliferative capacity of these regions. In the adult mammalian brain, the subventricular zone (SVZ) of the lateral ventricles is the main neurogenic niche. Neural stem/precursor cells (NSCs) within the SVZ play an important role in brain repair following injuries. However, the irradiated NSCs' ability to respond to damage has not been previously elucidated. In this study, we evaluated the effects of localized radiation on the SVZ ability to respond to a lysolecithin-induced demyelination of the striatum. We demonstrated that the proliferation rate of the irradiated SVZ was increased after brain damage and that residual NSCs were reactivated. The irradiated SVZ had an expansion of doublecortin positive cells that appeared to migrate from the lateral ventricles toward the demyelinated striatum, where newly generated oligodendrocytes were found. In addition, in the absence of demyelinating damage, remaining cells in the irradiated SVZ appeared to repopulate the neurogenic niche a year post-radiation. These findings support the hypothesis that NSCs are radioresistant and can respond to a brain injury, recovering the neurogenic niche. A more complete understanding of the effects that localized radiation has on the SVZ may lead to improvement of the current protocols used in the radiotherapy of cancer.

Effects of propentofylline on CNS remyelination in the rat brainstem.

Propentofylline (PPF) is a xanthine derivative with pharmacological effects distinct from those of the classical methylxanthines. It depresses activation of microglial cells and astrocytes which is associated with neuronal damage during neural inflammation and hypoxia. The aim of this study was to evaluate whether PPF had the capacity of affecting glial cells behavior during the process of demyelination and remyelination following ethidium bromide (EB) gliotoxic injury. EB injection into the CNS is commonly used as an experimental demyelinating model inducing local oligodendroglial and astrocytic death, which results in primary demyelination, blood-brain barrier and glia limitans disruption and Schwann cells invasion. Sixty Wistar rats were divided into four different groups receiving 10 microlitres of 0.1% EB or 0.9% saline solution into the cisterna pontis and treated or not with the xanthine. PPF treatment was done using 12.5 mg/kg/day by the intraperitonial route for 31 days of the experimental period. The rats were euthanized from 7 to 31 days after EB injection and brainstem sections were collected and processed for light and transmission electron microscopy studies. Results from both groups were compared by using a semi-quantitative method developed for documenting in semithin sections the extent and nature of remyelination of demyelinating lesions. Results showed that PPF administration after EB injection significantly increased both oligodendroglial and Schwann cell remyelination at 31 days (mean remyelination scores of 3.67 ± 0.5 for oligodendrocytes and 1.27 ± 0.49 for Schwann cells) compared to untreated animals (scores of 3.19 ± 0.57 and 0.90 ± 0.33, respectively).

The Notch signaling pathway: its role in focal CNS demyelination and apotransferrin-induced remyelination.

Oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Identifying the signaling pathways involved in myelin repair through oligodendroglial progenitor maturation is essential for the development of new therapies. This article investigated the role of the Notch signaling pathway in CNS demyelination and apotransferrin-induced remyelination in a focal lysolecithin-induced demyelination model in rats. Notch was found activated in Nestin-expressing neural progenitor cells and in NG2-expressing oligodendroglial precursor cells in the subventricular zone and corpus callosum of lysolecithin-demyelinated rats. Notch activation seemed to be driven by Jagged1, which led to a high expression of downstream gene Hes5 in the subventricular zone of demyelinated rats. Apotransferrin injection induced remyelination, while the injection of the γ-secretase inhibitor reversed this effect. In addition, 24 h after apotransferrin injection, evidence showed Notch activation concomitantly with an increase in F3/contactin levels and the up-regulation of the myelin-associated glycoprotein gene in the subventricular zone and corpus callosum of demyelinated rats. Collected evidence supports the participation of both canonical and non-canonical Notch signaling pathways in demyelination/remyelination. Notch activation was found to trigger Hes5 expression as a consequence of focal demyelination, which might promote oligodendroglial precursor cell proliferation. During apotransferrin-induced remyelination, Notch activation seemed to be mediated by the expression of F3/contactin, which might induce apotransferrin-mediated oligodendroglial maturation. Evidence of the participation of Notch signaling in the demyelination/remyelination process will help further understand demyelinating disorders such as Multiple Sclerosis and the use of aTf should be taken into consideration as a possible therapeutic intervention.

Sildenafil (Viagra) protective effects on neuroinflammation: the role of iNOS/NO system in an inflammatory demyelination model.

We recently demonstrated that sildenafil reduces the expression of cytokines, COX-2, and GFAP in a demyelinating model induced in wild-type (WT) mice. Herein, the understandings of the neuroprotective effect of sildenafil and the mediation of iNOS/NO system on inflammatory demyelination induced by cuprizone were investigated. The cerebella of iNOS(-/-) mice were examined after four weeks of treatment with cuprizone alone or combined with sildenafil. Cuprizone increased GFAP, Iba-1, TNF- α , COX-2, IL-1 ß , and IFN- γ expression, decreased expression of glutathione S-transferase pi (GSTpi), and damaged myelin in iNOS(-/-) mice. Sildenafil reduced Iba-1, IFN- γ , and IL-1 ß levels but had no effect on the expression of GFAP, TNF- α , and COX-2 compared to the cuprizone group. Sildenafil elevated GSTpi levels and improved the myelin structure/ultrastructure. iNOS(-/-) mice suffered from severe inflammation following treatment with cuprizone, while WT mice had milder inflammation, as found in the previous study. It is possible that inflammatory regulation through iNOS-feedback is absent in iNOS(-/-) mice, making them more susceptible to inflammation. Sildenafil has at least a partial anti-inflammatory effect through iNOS inhibition, as its effect on iNOS(-/-) mice was limited. Further studies are required to explain the underlying mechanism of the sildenafil effects.

Dopaminergic D2-like agonists produce yawning in the myelin mutant taiep and Sprague-Dawley rats.

Systemic administration of D2-like dopaminergic-receptor agonists increases yawning behavior. However, only a few studies have been done in animals with pathological conditions. The taiep rat is a myelin mutant with an initial hypomyelination followed by progressive demyelination, being the brainstem one of the most affected areas. In our experiments, we analyzed the effects of systemic administration of the D2-family agonists and antagonists on yawning behavior, and correlated them with the lipid myelin content in the brainstem and other areas in the central nervous system (CNS) in 8 month old male taiep and Sprague-Dawley rats. Subjects were maintained under standard conditions in Plexiglas cages with a 12:12 light-dark cycle, lights on at 0700 and free access to rodent pellets and tap water. Drugs were freshly prepared injected ip at 0800 and subjects were observed for 60 min. When antagonists were used it was administered 15 min before the agonist. Sprague-Dawley and taiep rats significantly increased their yawning frequency after systemic injection of (-)-quinpirole hydrochloride, R(+)-7-Hydroxy-2-(dipropylamino)tetralin hydrobromide (7-OH-DPAT) or trans-(±)-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano [4,3-b]-1,4-oxazin-9-ol hydrochloride ((±)-PD 128,907). Among D2-like agonists used higher effects are obtained with (-)-quinpirole. The effects caused by (-)-quinpirole can be reduced by (-)-sulpiride; and yawning caused by 7-OH-DPAT was decreased by tiapride only in taiep rats. In Sprague-Dawley only (-)-sulpiride is able to decrease (-)-quinpirole-caused yawning. In conclusion, dopaminergic D2-like agonists are still able to cause yawning despite the severe myelin loss in taiep rats. Similarly, patients with various CNS illnesses that affect myelin, such as stroke or multiple sclerosis, are able to yawn suggesting that trigger neurons are still able to command this innate behavior.

Cuprizone-induced demyelination in the rat cerebral cortex and thyroid hormone effects on cortical remyelination.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the Central Nervous System which is characterized by multifocal demyelinated lesions dispersed throughout the brain. Although white matter lesions have been the most extensively studied, cortical demyelinaton lesions are also detected in MS brains. Cuprizone (CPZ)-induced demyelination in rodents has been widely used as a model for MS. Most of these studies focus on oligodendrocyte-rich structures, such as the corpus callosum (CC) and the cerebellar peduncles. However, it has been recently described that CPZ administration in mice also produces cortical demyelination, resembling some of the lesions found in MS patients. In this work we used CPZ-demyelinating model in Wistar rats to study demyelination in cortical forebrain areas. At the ultrastructural level, demyelination in the cortex was observed before detectable myelin loss in the subcortical white matter. During the course of CPZ intoxication Myelin Basic Protein immunodetection was decreased in cortical layers I-III due to a reduction in the number of cortical oligodendrocytes (OL). Oligodendroglial loss in CPZ-intoxicated rats correlated with an increase in the number of Glial Fibrillary Acidic Protein positive astrocytes and a shift in the location of Carbonic Anhydrase II from OL to astrocytes. After removal of CPZ from the diet, we evaluate intranasal Thyroid hormone (TH) effects on the progression of cortical lesions. As previously reported in the CC, TH treatment also accelerates remyelination rate in the cortex compared to rats undergoing spontaneous remyelination. Our results suggest that manipulation of TH levels could be considered as a strategy to promote remyelination process in the cortex and to prevent neuronal irreversible damage in patients suffering from MS.

Adult CNP::EGFP transgenic mouse shows pronounced hypomyelination and an increased vulnerability to cuprizone-induced demyelination.

CNP::EGFP transgenic mice, genetically engineered to express the enhanced green fluorescent protein (EGFP) under the control of the 2-3-cyclic nucleotide 3-phosphodiesterase (CNPase) promoter in oligodendroglial and Schwann cells, constitute a very important and widely used tool for the study of oligodendrocyte (OLG) development and function in young mice. Our results showed that CNP::EGFP mice were significantly more susceptible to CPZ-induced demyelination, as evaluated by MBP immunostaining, oligodendroglial progenitor cell (OPC) recruitment and astroglial, microglial and nestin response. This enhanced vulnerability was a consequence of their hypomyelination. CNP::EGFP control mice also displayed a significant decrease in corpus callosum (CC) thickness and MBP immunoreactivity. Morphometric analysis further showed a significant decrease in the frequency of myelinated axons, myelin turns (lamellae) and g-ratio carried out in the optic nerve (ON) and CC of CNP::EGFP, as compared to WT mice. Moreover, our results showed a decrease in the number of axons of small caliber, concomitantly with an increase in the number of axons of bigger size with more and enlarged mitochondria, which suggests a high energy demand. These findings and those displaying that MBP+ cells and NF200 staining in the CNP::EGFP cortex were more sparsely distributed provide evidence of axonal loss, which was supported by a decreased number of NeuN+ cells in the CA3 fields of the hippocampus. Transgenic mice also showed an increase in microglial and astroglial activation, accompanied by enhanced lipid peroxidation and recruitment of morphologically altered OPC. Finally, CNPase protein levels proved to be lower than MBP in the CC, which might indicate an altered pattern in myelin proteins with a CNPase deficiency. Behavioral analysis of adult CNP::EGFP transgenic mice supported our results, as it revealed a decrease in locomotion, exploratory activity and motor impairment, as compared to their WT littermates. Our data highlight the relevance of confronting results obtained in adult CNP::EGFP mice with those observed in WT mice. According to our findings, CNP::EGFP hypomyelination might be triggered by the cellular stress induced by the high level of EGFP expression in mature OLG. Adult CNP::EGFP mice could be considered a useful tool to evaluate future therapies for demyelinating diseases such as multiple sclerosis (MS), since these animals present chronic demyelination with axonal degeneration, a characteristic of such pathologies.

Hemichannels in the neurovascular unit and white matter under normal and inflamed conditions.

In the normal brain, cellular types that compose the neurovascular unit, including neurons, astrocytes and endothelial cells express pannexins and connexins, which are protein subunits of two families that form plasma membrane channels. Most available evidence in mammals indicated that endogenously expressed pannexins only form hemichannels, and connexins form both gap junction channels and hemichannels. While gap junction channels connect the cytoplasm of contacting cells and coordinate electrical and metabolic activities, hemichannels communicate intra- and extracellular compartments and serve as diffusional pathways for ions and small molecules. Here, evidence supporting the functional role of hemichannels in the neurovascular unit and white matter under physiological and pathological conditions are reviewed. A sub-threshold acute pathological threatening condition (e.g., stroke and brain infection) leads to glial cell activation, which maintains an active defense and restores the normal function of the neurovascular unit. However, if the stimulus is deleterious, microglia and the endothelium become overactivated, both releasing bioactive molecules (e.g., glutamate, cytokines, prostaglandins and ATP) that increase the activity of astroglial hemichannels, reducing the astrocyte neuroprotective functions, and further reducing neuronal cell viability. Moreover, ATP is known to contribute to myelin degeneration of axons. Consequently, hemichannels might play a relevant role in the excitotoxic response of oligodendrocytes observed in ischemia and encephalomyelitis. Regulated changes in hemichannel permeability in healthy brain cells can have positive consequences in terms of paracrine/autocrine signaling, whereas persistent changes in cells affected by neurological disorders can be detrimental. Therefore, blocking hemichannels expressed by glial cells and/or neurons of the inflamed central nervous system might prevent neurovascular unit dysfunction and neurodegeneration.

Cuprizone-induced demyelination in CNP::GFP transgenic mice.

Cuprizone (bis-cyclohexanone oxaldihydrazone) was previously shown to induce demyelination in white matter enriched brain structures. In the present study we used the cuprizone demyelination model in transgenic mice expressing the enhanced green fluorescent protein (GFP) under the 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) promoter. The use of these particular transgenic mice allows easy detection of cells belonging to the entire oligodendroglial (OLG) lineage, ranging from OLG precursors to mature myelinating OLGs. We were able to evaluate the precise extent of oligodendroglial cell damage and recovery within the murine adult central nervous system (CNS) after inducing demyelination by acute cuprizone intoxication. A generalized loss of GFP+ cells was observed after cuprizone exposure and correlated with a decline in myelin basic protein (MBP) expression. OLGs were depleted in many brain areas that were previously thought to be unaffected by cuprizone treatment. Thus, in addition to the well-known cuprizone effects on the medial corpus callosum, we also found a loss of GFP+ cells in most brain structures, particularly in the caudatus putamen, cortex, anterior commissure, olfactory bulb, hippocampus, optic chiasm, brainstem, and cingulum. Loss of GFP+ cells was accompanied by extensive astrogliosis and microglial activation, although neurons were not affected. Interestingly, cuprizone-treated animals showed both activation of GFAP expression and a higher proliferation rate in subventricular zone cells. A week after cuprizone removal from the diet, GFP+ oligodendroglial cells began repopulating the damaged structures. GFP expression precedes that of MBP and allows OLG detection before myelin restoration.