Cordian Beyer

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.

IKK mediates ischemia-induced neuronal death

The IκB kinase complex IKK is a central component of the signaling cascade that controls NF-κB–dependent gene transcription. So far, its function in the brain is largely unknown. Here, we show that IKK is activated in a mouse model of stroke. To investigate the function of IKK in brain ischemia we generated mice that contain a targeted deletion of Ikbkb (which encodes IKK2) in mouse neurons and mice that express a dominant inhibitor of IKK in neurons. In both lines, inhibition of IKK activity markedly reduced infarct size. In contrast, constitutive activation of IKK2 enlarged the infarct size. A selective small-molecule inhibitor of IKK mimicked the effect of genetic IKK inhibition in neurons, reducing the infarct volume and cell death in a therapeutic time window of 4.5 h. These data indicate a key function of IKK in ischemic brain damage and suggest a potential role for IKK inhibitors in stroke therapy.

Nongenomic effects of oestrogen: embryonic mouse midbrain neurones respond with a rapid release of calcium from intracellular stores.

Evidence is emerging that oestrogen, besides acting via classical nuclear receptors, can rapidly influence the physiology of nerve cells through other mechanisms. Oestrogens have been shown to modulate the differentiation and function of embryonic midbrain dopaminergic neurones by stimulating neurite outgrowth, expression of tyrosine hydroxylase mRNA, dopamine uptake and release in spite of the fact that dopaminergic cells in the prenatal midbrain do not express the classical oestrogen receptor. This study therefore intended to unravel possible signal transduction pathways activated by oestrogen which might be associated with the above oestrogen effects. As a physiological second‐messenger mechanism, we studied the influence of oestrogen on fluctuations of intracellular Ca2+ levels [Ca2+]i by microspectrofluorimetry of the Ca2+‐sensitive indicator Fura‐2, in primary cultures from embryonic mouse midbrains. 17β‐estradiol (10 n m–1 p m) but not 17α‐estradiol increased [Ca2+]i within 1–3 s in a dose‐dependent way. Removal of extracellular Ca2+ abrogated K+‐stimulated Ca2+ rise but did not affect 17β‐estradiol stimulation. Pretreatment of cells with thapsigargin (1 μm, 10 min), an inhibitor of Ca2+‐pumping ATPases in the endoplasmic reticulum, abolished the 17β‐estradiol effect but not the K+‐stimulated [Ca2+]i rise. Oestrogen effects on [Ca2+]i were completely mimicked by using a membrane‐impermeant oestrogen‐BSA construct. In order to identify oestrogen‐sensitive cells, some cultures were subsequently immunostained for microtubule‐associated protein II, tyrosine hydroxylase, or GABA. All oestrogen‐sensitive cells were immunocytochemically characterized as neurones, and about half of these responsive neurones was found to be dopaminergic or GABAergic. These results demonstrate that 17β‐estradiol is capable of rapidly modulating physiological parameters of developing midbrain neurones by directly interacting with specific membrane binding sites coupled to a signal transduction mechanism that causes a calcium release from intracellular Ca2+ stores. It is suggested that oestrogen effects on differentiation and function of midbrain dopaminergic neurones are mediated by intracellular Ca2+ signalling.

Estrogenic Stimulation of Neurite Growth in Midbrain Dopaminergic Neurons Depends on cAMP/Protein Kinase A Signalling

Previous work from this laboratory indicates that the differentiation of mouse midbrain dopaminergic neurons is influenced by estrogen. These effects may be transmitted either through classical nuclear receptors or via “nongenomic” mechanisms, including the interaction with hypothetical membrane receptors coupled to distinct intracellular signalling pathways. The latter mechanism seems to be of particular interest for the observed interactions of estrogen with developing dopaminergic neurons, insofar as estrogen has been shown to increase intracellular calcium levels within seconds. This study focuses on signal transduction cascades that might be activated by estrogen during differentiation of dopaminergic cells. Treatment with 17β‐estradiol or a membrane‐impermeable estrogen‐BSA construct (E‐BSA) increased neurite growth and arborization of dopaminergic neurons. This effect was inhibited by antagonists of cAMP/protein kinase A (PKA) and calcium signalling pathways but not by the estrogen receptor antagonist ICI. In addition, estrogen exposure stimulated the phosphorylation of CREB in midbrain dopaminergic cells as studied by quantitative double‐labelling immunocytochemistry and gel shift assay. Again, this effect was antagonized only by the simultaneous treatment with inhibitors of the cAMP/PKA or calcium pathways and not by ICI pretreatment. These data together with our previous findings demonstrate that estrogen can interact with membrane binding sites on dopaminergic neurons, thereby stimulating the cAMP/PKA/phosphorylated cAMP‐responsive element binding protein (CREB) signalling cascade, most likely through the activation of calcium‐dependent kinases. In conclusion, rapid “nongenomic” estrogen signalling represents another mechanism, in addition to the activation of classical nuclear estrogen receptors, that is capable of influencing neuronal differentiation in the mammalian brain. J. Neurosci. Res. 59:107–116, 2000

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.

Expression of estrogen receptor-α and β mRNA in the developing and adult mouse striatum

Estrogen not only modulates nigrostriatal function but also developmental processes in the striatum. Recently, we have demonstrated the presence of the estrogen-synthesizing enzyme aromatase in the developing mouse striatum. This study is concerned with the expression of estrogen receptor-α/β (ER) mRNA in the developing and adult mouse striatum by semiquantitative reverse transcription-polymerase chain reaction. Expression of both ER subtypes occurred already prenatally and further increased until birth. Early postnatally, ER-α/β levels remained high but decreased to lower levels in adults. No sex difference in ER expression was observed. These data together with our previous findings demonstrate the simultaneous expression of both ER subtypes and aromatase in the mouse striatum. It is concluded that estrogen signalling through both nuclear receptors plays a potential role for striatal differentiation.

Ontogenetic expression and sex differences of aromatase and estrogen receptor-α/β mRNA in the mouse hippocampus.

Abstract. Estrogen plays an important role during brain development interfering with the maturation of distinct neural systems and, in particular, with the sexual differentiation of brain structures and function. Similar to other brain regions, estrogen is known to influence neuronal differentiation and plasticity in the hippocampus. The present study is concerned with the developmental expression of mRNAs for the estrogen-synthesizing enzyme aromatase and the two known nuclear estrogen receptors (α/β) in the male and female mouse hippocampus. Using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, we found that aromatase as well as estrogen receptors (α/β) are already expressed prenatally in the hippocampus of both sexes. Aromatase expression increased during the first two postnatal weeks and decreased, thereafter, to lower levels in adults. Sex differences in aromatase expression were observed postnatally with higher levels in males. Estrogen receptor-α/β mRNAs did not fluctuate obviously throughout pre- and postnatal development but revealed a distinct sex-specific pattern at the end of the first postnatal week. Again, higher expression was detected in males. These findings clearly demonstrate the capacity of estrogen formation and the presence of both estrogen receptor subtypes in the developing hippocampus. Sex differences in aromatase mRNA levels paralleled the sex-specific pattern of estrogen receptor expression. Thus, our data support the idea that the developing hippocampus is a target for estrogen action and estrogen receptor-mediated sexual differentiation.

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.

Membrane receptors for oestrogen in the brain

Oestrogen is important for the development of neuroendocrine centres and other neural networks including limbic and motor systems. Later in adulthood, oestrogen regulates the functional performance of different neural systems and is presumably implicated in the modulation of cognitive efficiency. Although still a matter of controversial discussion, clinical and experimental studies point at a potential neuroprotective role of oestrogen. Concerning the concept of cellular oestrogen action, it is undisputed that it comprises the binding and activation of nuclear receptors. The last decades have, however, immensely broadened the spectrum of steroid signalling within a cell. Novel steroid‐activated intracellular signalling mechanisms were described which are usually termed ‘non‐classical’ or ‘non‐genomic’. The brain appears to be a rich source of this new mode of oestrogen action. Studies from the past years have pinpointed non‐classical oestrogen effects in many CNS regions. All available data support the view that non‐classical oestrogen action requires interactions with putative membrane binding sites/receptors. In this article, we aim at compiling the most recent findings on the nature and identity of membrane oestrogen receptors with respect to the brain. We also attempt to turn readers attention to the coupling of these ‘novel’ receptors to distinct intracellular signalling pathways.