Cuprizone介导的急性脱髓动物模型髓鞘脱失及再生的机制

本研究通过在饲料中掺入cuprizone饲育小鼠,建立髓鞘可再生的急性脱髓动物模型,并利用髓鞘染色和原位杂交后免疫组化双标技术,检测髓鞘脱失和再生状况以及少突胶质前体细胞的改变。结果表明,给予cuprizone6周后,动物胼胝体严重脱髓鞘,少突胶质前体细胞在髓鞘脱失区域集聚,且增殖活跃;恢复正常饲料饲养4周后,髓鞘基本恢复正常形态。由此推测,在cuprizone介导的急性脱髓动物模型髓鞘脱失和再生过程中,少突胶质前体细胞的增殖活化为髓鞘再生提供了基础。     

cathepsin L及其抑制剂cystatin C在急性脱髓鞘动物模型中的表达及意义

在饲料中掺入cuprizone饲育小鼠6周,制备急性脱髓鞘动物模型。进而利用髓鞘荧光染色和原位杂交方法,检测动物胼胝体内髓鞘的脱失以及cathepsin L及其抑制剂cystatin C的表达情况。结果发现,与正常动物比较,cuprizone饲育6周后小鼠胼胝体内髓鞘脱失严重,胼胝体内cathepsin L和cystatin C的表达均显著上调。结果提示,髓鞘损伤后cathepsin L高表达可能有助于髓鞘残骸的清理,其抑制剂cystatin C的表达增强可将蛋白水解程度控制在一定范围内,这对髓鞘再生具有积极的意义。     

Mdivi-1对cuprizone诱导的脱髓鞘性病变小鼠少突胶质细胞的保护作用

目的:探讨线粒体分裂抑制剂1(Mdivi-1)对cuprizone诱导的脱髓鞘性病变模型小鼠少突胶质细胞损伤的作用及其机制。方法:将8周龄雄性C57BL/6小鼠用含0. 2%cuprizone的饲料饲喂,制备脱髓鞘性病变模型。将小鼠随机分为DMSO正常组(腹腔注射DMSO+饲喂正常饲料)、cuprizone模型组(饲喂含cuprizone的饲料)、DMSO+cuprizone模型组(腹腔注射DMSO+饲喂含cuprizone的饲料)和Mdivi-1干预组(腹腔注射Mdivi-1+饲喂含cuprizone的饲料)。通过固蓝染色及PLP、p-Drp1(Ser616)、CFB、C1q、C3b和C5b-9的免疫荧光染色实验检测Mdivi-1对髓鞘和少突胶质细胞的保护作用。结果:饲喂含cuprizone的饲料42 d可成功诱导小鼠脱髓鞘性病变模型,导致大脑胼胝体区髓鞘丢失,诱导Drp1磷酸化水平升高及C1q和CFB补体途径激活,并导致攻膜复合体在少突胶质细胞上组装。给予Mdivi-1可明显减少髓鞘丢失,抑制补体激活和攻膜复合体在少突胶质细胞的组装。结论:Mdivi-1干预可缓解cuprizone...     

神经小胶质细胞在EAE 脱髓鞘和髓鞘再生中的作用机制进展

炎性细胞浸润和脱髓鞘是中枢神经系统(CNS)的自身免疫性疾病---多发性硬化(MS)的主要病理特征,相关的病理研究多在其动物模型实验性自身免疫性脑脊髓膜炎(EAE)中开展。神经小胶质细胞(MG)是CNS 的主要免疫效应细胞,EAE 时它的激活在脱髓鞘和髓鞘再生中表现出复杂的作用。M1 型MG 是导致脱髓鞘的重要原因,抑制髓鞘再生;而M2型MG 可以促进髓鞘再生,抵抗脱髓鞘。本文综述MG 在EAE 脱髓鞘和髓鞘再生中的直接作用机制,及其通过星形胶质细胞产生的间接作用机制及进展。     

Cystatin F在中枢神经系统脱髓鞘动物模型中的表达

神经脱髓鞘疾病是中枢神经系统难治性疾病,髓鞘脱失后伴有各种基因表达的异常。明确这些基因的表达形式对脱髓鞘疾病的治疗具有重要意义。本研究利用0.2%cuprizone饲育C57BL/6小鼠6周制备中枢神经脱髓鞘模型,采用原位杂交技术对组织蛋白酶抑制剂cystatin F的mRNA表达进行研究。结果发现,正常野生型小鼠中枢神经系统中未见cystatin F mRNA的表达,而大量表达于脱髓鞘模型小鼠白质内。通过小胶质细胞标志物Iba1抗体免疫组化和c-fms的原位杂交研究发现,脱髓鞘模型小鼠白质内有大量激活的小胶质细胞。为进一步确定表达cystatin F的细胞类型,通过cystatin F原位杂交后Iba1免疫双染技术,确定cystatin F在中枢神经主要由激活的小胶质细胞表达,提示其表达与小胶质细胞活化和崩解髓鞘残骸清除有关。     

二氢丹参酮Ⅰ(DHTS1)通过抑制双环己酮草酰二腙诱导的小鼠小胶质细胞激活减轻髓鞘损伤

目的探讨二氢丹参酮I(DHTS1)对双环己酮草酰二腙(cuprizone)诱导脱髓鞘保护作用的机制。方法 DHTS1溶解于5 g/L羧甲基纤维素钠(CMC-Na)中,饲喂小鼠含2 g/L cuprizone的饲料制备脱髓鞘模型,给予DHTS1处理,实验动物随机分为CMC-Na正常组、 CMC-Na联合cuprizone处理组、 DHTS1联合cuprizone处理组。通过固蓝(LFB)组织化学染色、免疫荧光组织化学染色检测髓鞘碱性蛋白(MBP)和髓鞘蛋白脂(PLP)的表达判断髓鞘完整性,原位末端转移酶标记技术(TUNEL)检测细胞凋亡;免疫荧光组织化学染色检测小胶质细胞/巨噬细胞特异性蛋白钙离子结合接头蛋白分子1(Iba-1)、 CD86、 CD163的表达确定小胶质细胞极化情况。脂多糖(LPS)极化SIM-A9小胶质细胞, DHTS1处理,实验分为CMC-Na对照组、 DHTS1对照组、 CMC-Na联合LPS处理组和DHTS1联合LPS处理组。流式细胞术检测CD16/32、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)阳性细胞比例。结果与CMC-Na联合cuprizone模型组比较, DHTS1处理明显减少cuprizone诱导的小鼠大脑胼胝体区髓鞘脱失,减少细胞凋亡,减少Iba-1~+阿米巴样小胶质细胞的面积,减少CD86~+细胞数目,增加CD163~+细胞数目。与CMC-Na联合LPS处理组相比, DHTS1明显减少CD16/32~+、 TNF-α~+、 iNOS~+小胶质细胞的百分比。结论 DHTS1可以抑制cuprizone诱导的脱髓鞘和细胞凋亡,可能与DHTS1调节小胶质细胞极化,减轻中枢神经系统炎症反应有关。     

肌苷对CPZ介导的急性脱髓鞘小鼠行为学及皮质髓鞘的影响

目的:探讨肌苷对双环己酮草酰二腙(cuprizone,CPZ)介导的急性脱髓鞘小鼠行为学及皮质髓鞘的影响。方法:在普通饲料中掺入0.2%CPZ,饲养小鼠6 w,同时联合腹腔注射肌苷,制备脱髓鞘治疗模型,利用体重测量、Morris水迷宫、悬尾实验、透射电镜等技术,观察肌苷对脱髓鞘小鼠治疗后行为学及皮质髓鞘的影响。结果:(1)体重变化:与生理盐水对照组比较,CPZ损伤组、肌苷治疗组小鼠体重从第6 d开始均明显降低(P0.05);(2)行为学:Morris水迷宫空间定位实验中,与CPZ损伤组比较,肌苷治疗组平台象限停留时间明显增加(P0.05);悬尾实验中,小鼠6 min不动时间均无明显差异(P0.05);(3)皮质髓鞘电镜观察显示,肌苷治疗组小鼠的皮质髓鞘病理改变程度降低,有新生髓鞘。结论:通过含0.2%CPZ饲料饲养联合腹腔注射肌苷制备脱髓鞘治疗小鼠,可使小鼠的学习、记忆功能明显改善,皮质髓鞘有明显的修复与再生,揭示肌苷对伴有学习记忆功能障碍的脱髓鞘小鼠有髓鞘保护作用。     

Ski在大鼠活化星形胶质细胞中表达的变化

目的:探究Ski在大鼠正常及活化星形胶质细胞中的表达及随时间变化的规律。方法:提取大鼠大脑皮质,分离星形胶质细胞,体外培养。采用LPS刺激和细胞划痕损伤2种方法活化星形胶质细胞,均采用Western blot法检测各个时点2种活化星形胶质细胞中Ski和胶质纤维酸性蛋白(GFAP)蛋白表达情况,利用间接免疫荧光法检测Ski蛋白在星形胶质细胞中的表达定位。结果:GFAP蛋白在星形胶质细胞中固有表达,LPS活化和划痕损伤后表达均上调。Ski在正常星形胶质细胞中呈低表达状态,1 mg/L LPS活化星形胶质细胞后,Ski表达开始增加,4 d时表达量最高(P0.05),6 d时开始下降,但仍高于未活化组;细胞划痕损伤后Ski蛋白表达情况与上述情况高度一致。Ski在正常及LPS活化6 d星形胶质细胞中表达主要集中在胞核,LPS活化后2和4 d时在胞质中出现明确的Ski表达。结论:Ski蛋白表达于星形胶质细胞,并在活化星形胶质细胞中表达上调。由此,我们推测Ski蛋白可能调控星形胶质细胞的活化、增殖等过程。     

Ara-C处理小脑与异种视神经的联合体外培养研究

采用细胞分裂抑制剂阿糖胞苷(Cytosine Arabinoside,Ara-C)抑制体外培养小鼠小脑组织中少突胶质细胞的增殖而造成小脑组织的脱髓鞘模型,以10μg/ml阿糖胞苷处理7天为最佳剂量。利用该模型与大鼠视神经组织联合培养2周后,少突胶质细胞自视神经迁至经Ara-C处理的小脑组织中,并在视神经附近形成髓鞘,表明异种动物之间神经组织联合培养能形成髓鞘。少突胶质细胞在形成髓鞘前先进行分裂增殖。本实验建立和采用的体外小脑组织脱髓鞘模型和联合培养系统对研究影响中枢神经髓鞘再生的因素是有效的。     

从动物模型的视角研究多发性硬化髓鞘保护和再生的小胶质细胞靶点

小胶质细胞是定植于中枢神经系统(CNS)的免疫细胞,构成了CNS的第一道防线。多发性硬化(MS)是以炎症脱髓鞘伴轴突损伤为主要特征的CNS炎症变性疾病,小胶质细胞激活在其发生发展过程中担负着重要角色。在MS动物模型的CNS可见大量激活的小胶质细胞,其功能复杂,主要有促炎症M1表型和抗炎症M2表型两种小胶质细胞,具有破坏和保护髓鞘的双重作用:一方面M1表型小胶质细胞可通过释放促炎因子、自由基等对少突胶质细胞(OLs)及其前体细胞产生损伤,造成髓鞘破坏;另一方面M2表型小胶质细胞还可通过吞噬髓鞘碎片、分泌抗炎及再生因子等作用,加速髓鞘的修复和再生。本文对激活状态下M1/M2小胶质细胞的功能和靶向小胶质细胞转化在经典MS动物模型中的研究进展作一综述,为靶向小胶质细胞治疗CNS脱髓鞘疾病提供基础研究和临床应用的实验依据。     

Oligodendrocytes and progenitors become progressively depleted within chronically demyelinated lesions

To understand mechanisms that may underlie the progression of a demyelinated lesion to a chronic state, we have used the cuprizone model of chronic demyelination. In this study, we investigated the fate of oligodendrocytes during the progression of a demyelinating lesion to a chronic state and determined whether transplanted adult oligodendrocyte progenitors could remyelinate the chronically demyelinated axons. Although there is rapid regeneration of the oligodendrocyte population following an acute lesion, most of these newly regenerated cells undergo apoptosis if mice remain on a cuprizone diet. Furthermore, the oligodendrocyte progenitors also become progressively depleted within the lesion, which appears to contribute to the chronic demyelination. Interestingly, even if the mice are returned to a normal diet following 12 weeks of exposure to cuprizone, remyelination and oligodendrocyte regeneration does not occur. However, if adult O4+ progenitors are transplanted into the chronically demyelinated lesion of mice treated with cuprizone for 12 weeks, mature oligodendrocyte regeneration and remyelination occurs after the mice are returned to a normal diet. Thus, the formation of chronically demyelinated lesions induced by cuprizone appears to be the result of oligodendrocyte depletion within the lesion and not due to the inability of the chronically demyelinated axons to be remyelinated.     

Cortical demyelination is prominent in the murine cuprizone model and is strain-dependent

The cuprizone model of toxic demyelination in the central nervous system is commonly used to investigate the pathobiology of remyelination in the corpus callosum. However, in human demyelinating diseases such as multiple sclerosis, recent evidence indicates a considerable amount of cortical demyelination in addition to white matter damage. Therefore, we have investigated cortical demyelination in the murine cuprizone model. To induce demyelination, C57BL/6 mice were challenged with 0.2% cuprizone feeding for 6 weeks followed by a recovery phase of 6 weeks with a cuprizone-free diet. In addition to the expected demyelination in the corpus callosum, the cortex of C57BL/6 mice was completely demyelinated after 6 weeks of cuprizone feeding. After withdrawal of cuprizone the cortex showed complete remyelination similar to that in the corpus callosum. When C57BL/6 mice were fed cuprizone for a prolonged period of 12 weeks, cortical remyelination was significantly delayed. Because interstrain differences have been described, we also investigated the effects of cuprizone on cortical demyelination in BALB/cJ mice. In these mice, cortical demyelination was only partial. Moreover, cortical microglia accumulation was markedly increased in BALB/cJ mice, whereas microglia were absent in the cortex of C57BL/6 mice. In summary, our results show that cuprizone feeding is an excellent model in which to study cortical demyelination and remyelination, including contributing genetic factors represented by strain differences.     

Cerebellar Cortical Demyelination in the Murine Cuprizone Model

In multiple sclerosis, demyelination occurs beside the white‐matter structures and in the cerebral and cerebellar cortex. We have previously shown that, in the cuprizone model, demyelination is present not only in the corpus callosum but also in the cerebral cortex. Here, we have performed a detailed analysis of the dynamics of de‐ and remyelination in the cerebellar cortex and white matter at nine timepoints in two cerebellar regions. To induce demyelination, C57BL/6 mice were fed with 0.2% cuprizone for 12 weeks followed by a recovery of 8 weeks. Both cortex and white‐matter structures were significantly demyelinated after 12 weeks of cuprizone feeding. Remyelination occurred after withdrawal of cuprizone but was less prominent in the more caudal cerebellar region. Microglia infiltration was prominent in all analyzed cerebellar areas, preceding demyelination by approximately 2–4 weeks, and was delayed in the more caudal cerebellar region. Astrogliosis was also seen but did not reach the extent observed in the cerebrum. In summary, cuprizone feeding provides an excellent model for the investigation of de‐ and remyelination processes in the cerebellar cortex and white matter. Furthermore, demyelination, microglia and astrocyte changes were different in the cerebellum as compared with the cerebrum, indicating region‐dependent pathomechanisms.     

The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system

Myelin of the adult CNS is vulnerable to a variety of metabolic, toxic, and autoimmune insults. That remyelination can ensue, following demyelinating insult, has been well demonstrated. Details of the process of remyelination are, however difficult to ascertain since in most experimental models of demyelination/remyelination the severity, localization of lesion site, or time course of the pathophysiology is variable from animal to animal. In contrast, an experimental model in which massive demyelination can be reproducibly induced in large areas of mouse brain is exposure to the copper chelator, cuprizone, in the diet. We review work from several laboratories over the past 3 decades, with emphasis on our own recent studies, which suggest an overall picture of cellular events involved in demyelination/remyelination. When 8 week old C57BL/6 mice are fed 0.2% cuprizone in the diet, mature olidgodendroglia are specifically insulted (cannot fulfill the metabolic demand of support of vast amounts of myelin) and go through apoptosis. This is closely followed by recruitment of microglia and phagoctytosis of myelin. Studies of myelin gene expression, coordinated with morphological studies, indicate that even in the face of continued metabolic challenge, oligodendroglial progenitor cells proliferate and invade demyelinated areas. If the cuprizone challenge is terminated, an almost complete remyelination takes place in a matter of weeks. Communication between different cell types by soluble factors may be inferred. This material is presented in the context of a model compatible with present data -- and which can be tested more rigorously with the cuprizone model. The reproducibility of the model indicates that it may allow for testing of manipulations (e.g. available knockouts or transgenics on the common genetic background, or pharmacological treatments) which may accelerate or repress the process of demyelination and or remyelination.     

Chronic toxic demyelination in the central nervous system leads to axonal damage despite remyelination

The majority of multiple sclerosis lesions fail to remyelinate after chronic demyelinating episodes resulting in neurologic disability. In the current study, chronic demyelination was investigated by using the cuprizone model, a toxic demyelination model. C57BL/6 mice were administered a 0.2% cuprizone diet up to 16 weeks to induce chronic demyelination. For comparison, another group was maintained only for 6 weeks on cuprizone to model acute demyelination. Both groups were analysed regarding the remyelination process after withdrawal of the toxin. Reexpression of myelin proteins after chronic demyelination was reduced by a factor of two as judged by LFB and myelin protein stainings compared to acute demyelination after 2 weeks on remyelination. During chronic demyelination mature oligodendrocytes (Nogo-A positive cells) were severely depleted by 90% compared to age matched controls. Nevertheless, extensive remyelination occurred after withdrawal of cuprizone and was nearly complete after 12 weeks. There was only minimal acute axonal damage as judged by APP staining, with the course of APP positive axons correlating with macrophage/microglia accumulation. Chronic axonal damage detected by SMI-32 positive staining was only seen after chronic demyelination and was still observable during the whole remyelination period. These data suggest that two pattern of axonal injury occur in the cuprizone model.     

Deleterious role of IFNgamma in a toxic model of central nervous system demyelination

Interferon-gamma (IFNgamma) is a pleiotropic cytokine that plays an important role in many inflammatory processes, including autoimmune diseases such as multiple sclerosis (MS). Demyelination is a hallmark of MS and a prominent pathological feature of several other inflammatory diseases of the central nervous system, including experimental autoimmune encephalomyelitis, an animal model of MS. Accordingly, in this study we followed the effect of IFNgamma in the demyelination and remyelination process by using an experimental autoimmune encephalomyelitis model of demyelination/remyelination after exposure of mice to the neurotoxic agent cuprizone. We show that demyelination in response to cuprizone is delayed in mice lacking the binding chain of IFNgamma receptor. In addition, IFNgammaR(-/-) mice exhibited an accelerated remyelination process after cuprizone was removed from the diet. Our results also indicate that the levels of IFNgamma were able to modulate the microglia/macrophage recruitment to the demyelinating areas. Moreover, the accelerated regenerative response showed by the IFNgammaR(-/-) mice was associated with a more efficient recruitment of oligodendrocyte precursor cells in the demyelinated areas. In conclusion, this study suggests that IFNgamma regulates the development and resolution of the demyelinating syndrome and may be associated with toxic effects on both mature oligodendrocytes and oligodendrocyte precursor cells.     

Functional recovery of callosal axons following demyelination: a critical window

Axonal dysfunction as a result of persistent demyelination has been increasingly appreciated as a cause of functional deficit in demyelinating diseases such as multiple sclerosis. Therefore, it is crucial to understand the ultimate causes of ongoing axonal dysfunction and find effective measures to prevent axon loss. Our findings related to functional deficit and functional recovery of axons from a demyelinating insult are important preliminary steps towards understanding this issue. Cuprizone diet for 3–6 wks triggered extensive corpus callosum (CC) demyelination, reduced axon conduction, and resulted in loss of axon structural integrity including nodes of Ranvier. Replacing cuprizone diet with normal diet led to regeneration of myelin, but did not fully reverse the conduction and structural deficits. A shorter 1.5 wk cuprizone diet also caused demyelination of the CC, with minimal loss of axon structure and nodal organization. Switching to normal diet led to remyelination and restored callosal axon conduction to normal levels. Our findings suggest the existence of a critical window of time for remyelination, beyond which demyelinated axons become damaged beyond the point of repair and permanent functional loss follows. Moreover, initiating remyelination early within the critical period, before prolonged demyelination-induced axon damage ensues, will improve functional axon recovery and inhibit disease progression.     

Early regional cuprizone‐induced demyelination in a rat model revealed with MRI

The cuprizone model of demyelination is well established in the mouse as a tool for the study of the mechanisms of both demyelination and remyelination. It is often desirable, however, to have a larger model, such as the rat, especially for imaging‐based studies, yet initial work has failed to show demyelination in cuprizone‐fed rats. Several recent studies have demonstrated demyelination in the rat, but only in the corpus callosum. In this study, we acquired high‐resolution, three‐dimensional images of the whole brain every 2 weeks, using a T₁‐weighted magnetization‐prepared rapid acquisition gradient echo imaging sequence, optimized for myelin contrast, in order to assess myelination across the entire rat brain over a period of 8 weeks on a 1% cuprizone diet. We observed a consistent pattern of demyelination, beginning in the cerebellum by 4 weeks and involving more rostral regions of the brain by 8 weeks on the cuprizone diet, with validation using Luxol fast blue histology. This imaging technique permits the effects of cuprizone‐induced demyelination to be followed longitudinally in a single animal, over the entire brain. In turn, this may facilitate the establishment of the cuprizone model of demyelination in the rat.