Markus Kipp

The cuprizone animal model: new insights into an old story

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease that affects the central nervous system and represents the most common neurological disorder in young adults in the Western hemisphere. There are several well-characterized experimental animal models that allow studying potential mechanisms of MS pathology. While experimental allergic encephalomyelitis is one of the most frequently used models to investigate MS pathology and therapeutic interventions, the cuprizone model reflects a toxic experimental model. Cuprizone-induced demyelination in animals is accepted for studying MS-related lesions and is characterized by degeneration of oligodendrocytes rather than by a direct attack on the myelin sheet. The present article reviews recent data concerning the cuprizone model and its relevance for MS. Particular focus is given to the concordance and difference between human MS patterns (types I–IV lesions) and cuprizone-induced histopathology, including a detailed description of the sensitive brain regions extending the observations to different white and grey matter structures. Similarities between pattern III lesions and cuprizone-induced demyelination and dissimilarities, such as inflamed blood vessels or the presence of CD3+ T cells, are outlined. We also aim to distinguish acute and chronic demyelination under cuprizone including processes such as spontaneous remyelination during acute demyelination. Finally, we point at strain and gender differences in this animal model and highlight the contribution of some growth factors and cytokines during and after cuprizone intoxication, including LIF, IGF-1, and PDGFα.

Astrocytes regulate myelin clearance through recruitment of microglia during cuprizone-induced demyelination

Recent evidence suggests that astrocytes play an important role in regulating de- and remyelination in multiple sclerosis. The role of astrocytes is controversial, and both beneficial as well as detrimental effects are being discussed. We performed loss-of-function studies based on astrocyte depletion in a cuprizone-induced rodent model of demyelination. This led to strong astrogliosis accompanied by microgliosis and demyelination in C57BL/6 wild-type mice. Ablation of astrocytes in glial fibrillary acidic protein-thymidine kinase transgenic mice was associated with a failure of damaged myelin removal and a consecutive delay in remyelination. Despite oligodendrocyte death, myelin was still present, but ultrastructual investigations showed that the myelin structure was loosened and this damaged myelin did not protect axons. These alterations were associated with a decrease in microglial activation. Thus, our results show that astrocyte loss does not prevent myelin damage, but clearance of damaged myelin through recruitment of microglia is impaired. Further studies suggest that this process is regulated by the chemokine CXCL10. As a consequence of the delayed removal of myelin debris, remyelination and oligodendrocyte precursor cell proliferation were impaired. Experiments omitting the influence of myelin debris demonstrated an additional beneficial effect of astrocytes on oligodendrocyte regeneration during remyelination. In conclusion, these data demonstrate for the first time in vivo that astrocytes provide the signal environment that forms the basis for the recruitment of microglia to clear myelin debris, a process required for subsequent repair mechanisms. This is of great importance to understanding regenerative processes in demyelinating diseases such as multiple sclerosis.

17β-Estradiol and progesterone prevent cuprizone provoked demyelination of corpus callosum in male mice

Sex hormones, for example, estrogen and progesterone, are thought to affect and delay progression of multiple sclerosis (MS) in pregnant women. Although both steroid hormones are neuroprotective in the brain and elevated during pregnancy, only estrogen was tested in clinical trials. To evaluate the role of 17β‐estradiol (E) and progesterone (P) in prevention demyelination, young adult male mice were fed with cuprizone for a defined time interval and simultaneously treated with steroids by repeated injections into the neck region. The status of myelination was analyzed by magnetic resonance imaging and conventional histological staining. The individual application of E and P resulted only in a moderate prevention of demyelination in the corpus callosum (CC). The combined treatment with both steroid hormones counteracted the process of demyelination. Expression of the mature (PLP and MBP) and premature (PDGF‐α‐R) oligodendrocyte markers were significantly increased after hormone application in the affected CC. In addition, both hormones stimulated astrogliosis and the expression of IGF‐1. Microglial invasion in demyelinated CC was pronounced and additionally localized in the midline of CC after hormone treatment. These data show that sex steroids can protect the brain from demyelination and stimulate remyelination. It appears that only the administration of both hormones is fully effective. The beneficial steroid effect requires interactions with oligodendrocytes possibly by preventing their degeneration or recruitment from precursor cells which are stimulated to remyelinated fibers. The positive hormonal influence on myelination in the CNS may be a future therapeutically strategy for the treatment of MS.

Gonadal steroids prevent cell damage and stimulate behavioral recovery after transient middle cerebral artery occlusion in male and female rats

17β-estradiol (E) and progesterone (P) are neuroprotective factors in the brain preventing neuronal death under different injury paradigms. Our previous work demonstrates that both steroids compensate neuronal damage and activate distinct neuroprotective strategies such as improving local energy metabolism and abating pro-inflammatory responses. The current study explored steroid hormone-mediated protection from brain damage and restoration of behavioral function after 1h transient middle cerebral artery occlusion (tMCAO). Male and ovariectomized female rats were studied 24h after stroke. Both steroid hormones reduced the cortical infarct area in males and females to a similar extent. A maximum effect of ~60-70% reduction of the infarct size was evident after P and a combined treatment with both hormones. No infarct protection was seen in the basal ganglia. Testing of motor and sensory behavioral revealed an equal high degree of functional recovery in all three hormone groups. Gene expression studies in the delineated penumbra revealed that estrogen receptor (ER) alpha and beta are locally up-regulated. tMCAO-mediated induction of the pro-inflammatory chemokines CCL2, CCL5 and interleukin 6 was attenuated by E and P, whereas the expression of vascular endothelial growth factor (VEGF) was fortified. Local expression of microglia/macrophage/lymphocyte markers, i.e. Iba1, CD68 and CD3, were significantly reduced in the penumbra after hormone treatment suggesting attenuation of microglia and lymphocyte attraction. These results demonstrate the neuroprotective potency of a combined treatment with E and P under ischemic conditions in both sexes and point at the regulation of chemokine-microglia/lymphocyte interactions as a supposable mechanism implicated in cell protection.

Impact of sex steroids on neuroinflammatory processes and experimental multiple sclerosis

Synthetic and natural estrogens as well as progestins modulate neuronal development and activity. Neurons and glia are endowed with high-affinity steroid receptors. Besides regulating brain physiology, both steroids conciliate neuroprotection against toxicity and neurodegeneration. The majority of data derive from in vitro studies, although more recently, animal models have proven the efficaciousness of steroids as neuroprotective factors. Indications for a safeguarding role also emerge from first clinical trials. Gender-specific prevalence of degenerative disorders might be associated with the loss of hormonal activity or steroid malfunctions. Our studies and evidence from the literature support the view that steroids attenuate neuroinflammation by reducing the pro-inflammatory property of astrocytes. This effect appears variable depending on the brain region and toxic condition. Both hormones can individually mediate protection, but they are more effective in cooperation. A second research line, using an animal model for multiple sclerosis, provides evidence that steroids achieve remyelination after demyelination. The underlying cellular mechanisms involve interactions with astroglia, insulin-like growth factor-1 responses, and the recruitment of oligodendrocytes.

Sulforaphane suppresses LPS-induced inflammation in primary rat microglia

Objective and designThe aim of this study was to investigate the signal transduction pathways involved in sulforaphane (SF) mediated inhibition of the inflammatory response to lipopolysaccharide (LPS). Additionally, we investigated the effects of SF and LPS on the activity of Nrf2.MaterialPrimary rat microglia and the murine microglia cell line BV2 were used.TreatmentCells were treated with LPS with or without SF.MethodsCell viability was measured via WST-assay. Real-time RT-PCR was performed to analyze cytokine mRNA levels. The nitric oxide (NO) release was measured in LPS-stimulated microglia. The induction of various signal transduction pathways and Nrf2 was determined by Western blotting. NF-κB and AP-1 activation was measured by dual luciferase assay.ResultsWe showed that SF attenuates the LPS-induced increase of IL-1β, IL-6, and TNF-α expression in microglia. In addition, SF significantly decreases the NO in a concentration-dependent manner. SF inhibits LPS-stimulated ERK1/2 and JNK phosphorylation and thereby inhibits the LPS-induced activation of NF-κB- and activator protein-1 (AP-1). Moreover, SF and LPS together are able to induce Nrf2 activation.ConclusionsWe showed that SF, and also LPS by itself, are able to activate the cell’s defence against oxidative and electrophilic stress. We conclude that SF could be a candidate agent for anti-inflammatory treatment of the central nervous system.

Estrogen and the development and protection of nigrostriatal dopaminergic neurons: Concerted action of a multitude of signals, protective molecules, and growth factors

The nigrostriatal dopamine system comprises the dopaminergic neurons located in the ventral midbrain, their axonal connections to the forebrain, and their direct cellular target cells in the striatal complex, i.e. GABAergic neurons. The major function of the nigrostriatal dopaminergic unit is the coordination and fine tuning of motor functions at the extrapyramidal level. Numerous biologically active factors including different types of growth factors (neurotrophins, members of the TGFbeta family, IGFs) and peptide/steroid hormones have been identified in the past to be implicated in the regulation of developmental aspects of this neural system. Some of these developmentally active determinants have in addition been found to play a crucial role in the mediation of neuroprotection concerning dopaminergic neurons. Estrogen was identified as such a compound interfering with embryonic neuronal differentiation and cell survival. The physiological mechanisms underlying these effects are very complex and include interactions with other developmental signals (growth factors), inflammatory processes as well as apoptotic events, but also require the activation of nonneuronal cells such as astrocytes. It appears that estrogen is assuming control over or at least influences a multitude of developmental and protective cellular mechanisms rather than taking over the part of a singular protagonist.

Myelin debris regulates inflammatory responses in an experimental demyelination animal model and multiple sclerosis lesions

In multiple sclerosis (MS), gray matter pathology is characterized by less pronounced inflammation when compared with white matter lesions. Although regional differences in the cytoarchitecture may account for these differences, the amount of myelin debris in the cortex during a demyelinating event might also be contributory. To analyze the association between myelin debris levels and inflammatory responses, cortical areas with distinct and sparse myelination were analyzed for micro‐ and astrogliosis before and after cuprizone‐induced demyelination in mice. In postmortem tissue of MS patients, leucocortical lesions were assessed for the type and level of inflammation in the cortical and white matter regions of the lesion. Furthermore, mice were injected intracerebrally with myelin‐enriched debris, and the inflammatory response analyzed in white and grey matter areas. Our studies show that the magnitude of myelin loss positively correlates with microgliosis in the cuprizone model. In MS, the number of MHC class II expressing cells is higher in the white compared with the grey matter part of leucocortical lesions. Finally, direct application of myelin debris into the corpus callosum or cortex of mice induces profound and comparable inflammation in both regions. Our data suggest that myelin debris is an important variable in the inflammatory response during demyelinating events. Whether myelin‐driven inflammation affects neuronal integrity remains to be clarified.

In vitro and in vivo models of multiple sclerosis

Multiple sclerosis (MS) is widely considered to be the result of an aggressive autoreactive T cell attack on myelin. How these autoimmune responses arise in MS is unclear, but they could result from virus infections. Thus, viral and autoimmune diseases in animals have been used to investigate the possible pathogenic mechanisms operating in MS. The autoimmune model, experimental autoimmune encephalomyelitis, is the most widely-used animal model and has greatly influenced therapeutic approaches targeting autoimmune responses. To investigate demyelination and remyelination in the absence of the adaptive immune response, toxin-induced demyelination models are used. These include using cuprizone, ethidium bromide and lysolecithin to induce myelin damage, which rapidly lead to remyelination when the toxins are withdrawn. The virus models include natural and experimental infections such as canine distemper, visna infection of sheep, and infection of non-human primates. The most commonly used viral models in rodents are Semliki Forest virus and Theilers murine encephalomyelitis virus. The viral and experimental autoimmune encephalomyelitis models have been instrumental in the understanding of how viruses trigger inflammation, demyelination and neurodegeneration in the central nervous system. However, due to complexity of the animal models, pathological mechanisms are also examined in central nervous system cell culture systems including co-cultures, aggregate cultures and brain slice cultures. Here we critically review in vitro and in vivo models used to investigate MS. Since knowledge gained from these models forms the basis for the development of new therapeutic approaches for MS, we address the applicability of the models. Finally, we provide guidance for using and reporting animal studies with the aim of improving translational studies to the clinic.

Oligodendrocyte-microglia cross-talk in the central nervous system

Communication between the immune system and the central nervous system (CNS) is exemplified by cross‐talk between glia and neurons shown to be essential for maintaining homeostasis. While microglia are actively modulated by neurons in the healthy brain, little is known about the cross‐talk between oligodendrocytes and microglia. Oligodendrocytes, the myelin‐forming cells in the CNS, are essential for the propagation of action potentials along axons, and additionally serve to support neurons by producing neurotrophic factors. In demyelinating diseases such as multiple sclerosis, oligodendrocytes are thought to be the victims. Here, we review evidence that oligodendrocytes also have strong immune functions, express a wide variety of innate immune receptors, and produce and respond to chemokines and cytokines that modulate immune responses in the CNS. We also review evidence that during stress events in the brain, oligodendrocytes can trigger a cascade of protective and regenerative responses, in addition to responses that elicit progressive neurodegeneration. Knowledge of the cross‐talk between microglia and oligodendrocytes may continue to uncover novel pathways of immune regulation in the brain that could be further exploited to control neuroinflammation and degeneration.