Tim Clarner

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.

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.

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.

Phagocytosis of neuronal debris by microglia is associated with neuronal damage in multiple sclerosis.

Neuroaxonal degeneration is a pathological hallmark of multiple sclerosis (MS) contributing to irreversible neurological disability. Pathological mechanisms leading to axonal damage include autoimmunity to neuronal antigens. In actively demyelinating lesions, myelin is phagocytosed by microglia and blood‐borne macrophages, whereas the fate of degenerating or damaged axons is unclear. Phagocytosis is essential for clearing neuronal debris to allow repair and regeneration. However, phagocytosis may lead to antigen presentation and autoimmunity, as has been described for neuroaxonal antigens. Despite this notion, it is unknown whether phagocytosis of neuronal antigens occurs in MS. Here, we show using novel, well‐characterized antibodies to axonal antigens, that axonal damage is associated with HLA‐DR expressing microglia/macrophages engulfing axonal bulbs, indicative of axonal damage. Neuronal proteins were frequently observed inside HLA‐DR+ cells in areas of axonal damage. In vitro, phagocytosis of neurofilament light (NF‐L), present in white and gray matter, was observed in human microglia. The number of NF‐L or myelin basic protein (MBP) positive cells was quantified using the mouse macrophage cell line J774.2. Intracellular colocalization of NF‐L with the lysosomal membrane protein LAMP1 was observed using confocal microscopy confirming that NF‐L is taken up and degraded by the cell. In vivo, NF‐L and MBP was observed in cerebrospinal fluid cells from patients with MS, suggesting neuronal debris is drained by this route after axonal damage. In summary, neuroaxonal debris is engulfed, phagocytosed, and degraded by HLA‐DR+ cells. Although uptake is essential for clearing neuronal debris, phagocytic cells could also play a role in augmenting autoimmunity to neuronal antigens.

Cuprizone treatment induces distinct demyelination, astrocytosis, and microglia cell invasion or proliferation in the mouse cerebellum

Demyelination of the cerebellum is a well-known phenomenon in human multiple sclerosis (MS). Concordantly, patients with MS frequently developed symptoms deriving from cerebellar lesions, i.e., dysmetria leading to hand dexterity impairment. Important advances in MS research have been made as a direct or indirect consequence of the establishment of adequate animal models. In this study, we used the cuprizone mouse model to investigate cerebellar demyelination in young adult male mice. The myelin status was analyzed by immunohistochemistry for proteolipoprotein and electron microscopy. The expression and presence of oligodendrocyte, astroglial, and microglia markers were supplementary studied. Cuprizone intoxication induced an almost complete demyelination of cerebellar nuclei. Cerebellar cortex regions were not (cortical gray matter) or only marginally (cortical white matter) affected. In addition, the affected areas displayed hypertrophic and hyperplastic astrocytosis accompanied by microglia or macrophage invasion. We conclude that cuprizone-induced demyelination pictures cerebellar deep gray matter involvement but not cerebellar cortex pathology as described for human MS. Behavioral changes after cuprizone described for this animal model may not only result from effects on commissural fiber tracts but also can arise from cerebellar demyelination.

Cuprizone treatment induces demyelination and astrocytosis in the mouse hippocampus

Memory impairment is outstanding within the spectrum of cognitive deficits in multiple sclerosis (MS) patients. Demyelination has been reported in the hippocampus formation of MS patients. The degree of hippocampus lesions in MS strongly correlates with progression of cognitive dysfunction. Because no appropriate animal model for the study of hippocampus demyelination has been established, we used the cuprizone mouse model to investigated demyelination in young adult and aged mice. The myelin status was analyzed by classical histological staining, immunocytochemistry for proteolipoprotein, and electron microscopy. Oligodendrocyte, astroglial, and microglia markers were studied. Cuprizone intoxication induced an almost complete demyelination of distinct hippocampus subregions to a similar extent in young adult and aged male mice. Demyelination was pronounced in a subset of white and gray matter areas, i.e., the stratum lacunosum moleculare containing the perforant path, medial alveus, stratum pyramidale in the cornu ammonis 2/3 region, and hilus region. Besides demyelination, affected areas displayed hypertrophic and hyperplastic astrocytosis. No significant effect on microglia invasion was detected at any investigated time point (0, 3, 5, and 7 weeks). We conclude that cuprizone‐induced demyelination provides an adequate animal model to investigate appropriate therapy strategies for the prevention of hippocampus demyelination.

TTC staining of damaged brain areas after MCA occlusion in the rat does not constrict quantitative gene and protein analyses.

In models of ischemic stroke, TTC (2,3,5-triphenyltetrazolium chloride) staining is commonly applied for the fast and reliable visualization of hypoxic brain tissue and for defining the size of cerebral infarction and penumbra. Deciphering molecular processes of pathogenesis within the penumbra is of particular interest for the development of therapeutic strategies. The aim of this study was to assess whether TTC-stained tissues can easily and in a reliable quantitative manner be processed for further molecular and biochemical analyses. We applied phenol-based RNA isolation, protein lysis by conventional RIPA buffer, and combined RNA/protein isolation with NucleoSpinRNA/Protein-Kit. Gene and protein expression analyses were performed by RT-rtPCR and Western-blotting. Middle cerebral arteria occlusion (MCAO) in rats was performed following a standardized experimental procedure. After MCAO, TTC staining revealed massive cell death in cortical and sub-cortical areas. TTC processing did not affect the quality of tissue RNA and protein. The expression of housekeeping and regulatory genes and proteins revealed no difference between control and TTC-stained groups. The expression of known stroke-regulated genes such as TNFalpha and IL1beta revealed similar induction profiles after TTC staining as described in the literature. TTC staining allows the precise delineation of lesioned and primarily non-lesioned brain areas for subsequent dissection of selected tissue pieces for molecular analysis. Our study demonstrates that TTC-stained tissues in stroke animal models can be used for quantitative gene and protein expression analyses without constriction. Pathomechanisms of ongoing tissue damage within the penumbra region can now be investigated in detail.

Inflammatory Response and Chemokine Expression in the White Matter Corpus Callosum and Gray Matter Cortex Region During Cuprizone-Induced Demyelination

Brain inflammation plays a central role in multiple sclerosis (MS). Besides lymphocytes, the astroglia and microglia mainly contribute to the cellular composition of the inflammatory infiltrate in MS lesions. Several studies were able to demonstrate that cortical lesions are characterized by lower levels of inflammatory cells among activated microglia/macrophages. The underlying mechanisms for this difference, however, remain to be clarified. In the current study, we compared the kinetics and extent of microglia and astrocyte activation during early and late cuprizone-induced demyelination in the white matter tract corpus callosum and the telencephalic gray matter. Cellular parameters were related to the expression profiles of the chemokines Ccl2 and Ccl3. We are clearly able to demonstrate that both regions are characterized by early oligodendrocyte stress/apoptosis with concomitant microglia activation and delayed astrocytosis. The extent of microgliosis/astrocytosis appeared to be greater in the subcortical white matter tract corpus callosum compared to the gray matter cortex region. The same holds true for the expression of the key chemokines Ccl2 and Ccl3. The current study defines a model to study early microglia activation and to investigate differences in the neuroinflammatory response of white vs. gray matter.

BLBP-expression in astrocytes during experimental demyelination and in human multiple sclerosis lesions

Several lines of evidence indicate that remyelination represents one of the most effective mechanisms to achieve axonal protection. For reasons that are not yet understood, this process is often incomplete or fails in multiple sclerosis (MS). Activated astrocytes appear to be able to boost or inhibit endogenous repair processes. A better understanding of remyelination in MS and possible reasons for its failure is needed. Using the well-established toxic demyelination cuprizone model, we created lesions with either robust or impaired endogenous remyelination capacity. Lesions were analyzed for mRNA expression levels by Affymetrix GeneChip® arrays. One finding was the predominance of immune and stress response factors in the group of genes which were classified as remyelination-supporting factors. We further demonstrate that lesions with impaired remyelination capacity show weak expression of the radial-glia cell marker brain lipid binding protein (BLBP, also called B-FABP or FABP7). The expression of BLBP in activated astrocytes correlates with the presence of oligodendrocyte progenitor cells. BLBP-expressing astrocytes are also detected in experimental autoimmune encephalomyelitis during the remission phase. Furthermore, highest numbers of BLBP-expressing astrocytes were evident in lesions of early MS, whereas significantly less are present at the rim of (chronic)-active lesions from patients with long disease duration. Transfection experiments show that BLBP regulates growth factor expression in U87 astrocytoma cells. In conclusion, we provide evidence that expression of BLBP in activated astrocytes negatively correlates with disease duration and in parallel with remyelination failure.

Cuprizone effect on myelination, astrogliosis and microglia attraction in the mouse basal ganglia.

Multiple sclerosis is the leading cause of neurological disability in young adults affecting more than two million people worldwide. Although multiple sclerosis is generally considered as white matter disease, distinct pathological alterations are also found in the grey matter. Involvement of basal ganglia seems to be related to a set of symptoms such as fatigue, impaired cognition, and movement disturbance. Since no appropriate animal model for studying cortical deep grey matter demyelination is established, we reassessed the cuprizone mouse model to investigate basal ganglia demyelination. Mice were fed cuprizone for different time intervals. The myelin status was analyzed by classical histological staining and immunohistochemistry for myelin proteins and glia markers. Expression of oligodendrocyte and astroglia were investigated by PCR. Cuprizone intoxication induced a severe demyelination of distinct cortical deep grey matter sub-regions. Striosmomes, located within the caudate-putamen and the ventral part of the caudate nucleus displayed intense demyelination, whereas those within the globus pallidus and the head of the caudate nucleus were not affected. The matrix region, however, was equally affected in the medial and lateral region. Besides demyelination, we observed hypertrophic and hyperplastic astrocytosis and microglia cell invasion/local proliferation in the demyelinated areas. Young adult and aged mice were similarly affected as well as mice with different genetic backgrounds. We conclude that cuprizone-induced demyelination provides an adequate animal model to investigate appropriate therapy strategies for the prevention of cortical deep grey matter demyelination. The heterogeneity in local demyelination points at beginning remyelination during ongoing demyelination.