Jon Dang

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.

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.

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.

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.

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.

Selective regulation of growth factor expression in cultured cortical astrocytes by neuro-pathological toxins

Astrocytes are integrated in the complex regulation of neurodegeneration and neuronal damage in the CNS. It is well-known that astroglia produces a plethora of growth factors which might be protective for neurons. Growth factors prevent neurons from cell death and promote proliferation and differentiation of precursor cells. Previous data suggest that astrocytes may respond to toxic stimuli by a selective mobilization of guarding molecules. In the present study, we have investigated the potency of different pathological stimuli such as lipopolysaccharides, tumor necrosis factor alpha, glutamate, and hydrogen peroxide to activate cultured cortical astroglia and stimulate growth factor expression. Astroglial cultures were exposed to the above factors for 24h at non-toxic concentrations for astrocytes. Growth factor expression was analyzed by real-time PCR, oligo-microarray technique, and ELISA. Insulin-like growth factor-1 was selectively down-regulated by lipopolysaccharides and tumor necrosis factor alpha, bone morphogenetic protein 6 by all stimuli. In contrast, lipopolysaccharides, tumor necrosis factor alpha, and glutamate increased leukemia inhibitory factor. Fibroblast growth factor 2 was up-regulated by lipopolysaccharides and tumor necrosis factor alpha and down-regulated by hydrogen peroxide. Besides hydrogen peroxide, all other stimuli promoted vascular epithelial growth factor A mRNA and protein expression. It appears that lipopolysaccharides but not tumor necrosis factor alpha effects on vascular epithelial growth factor A depend on the classic NFkappaB pathway. Our data clearly demonstrate that astroglia actively responses to diverse pathological compounds by a selective expression pattern of growth factors. These findings make astrocytes likely candidates to participate in disease-specific characteristics of neuronal support or damage.

Omega-3 polyunsaturated fatty acids ameliorate neuroinflammation and mitigate ischemic stroke damage through interactions with astrocytes and microglia

Omega-3 polyunsaturated fatty acids (PUFA n3) provide neuroprotection due to their anti-inflammatory and anti-apoptotic properties as well as their regulatory function on growth factors and neuronal plasticity. These qualities enable PUFA n3 to ameliorate stroke outcome and limit neuronal damage. Young adult male rats received transient middle cerebral artery occlusion (tMCAO). PUFA n3 were intravenously administered into the jugular vein immediately after stroke and 12h later. We analyzed stroke volume and behavioral performance as well as the regulation of functionally-relevant genes in the penumbra. The extent of ischemic damage was reduced and behavioral performance improved subject to applied PUFA n3. Expression of Tau and growth-associated protein-43 genes were likewise restored. Ischemia-induced increase of cytokine mRNA levels was abated by PUFA n3. Using an in vitro approach, we demonstrate that cultured astroglial and microglia directly respond to PUFA n3 administration by preventing ischemia-induced increase of cyclooxygenase 2, hypoxia-inducible factor 1alpha, inducible nitric oxide synthase, and interleukin 1beta. Cultured cortical neurons also appeared as direct targets, since PUFA n3 shifted the Bcl-2-like protein 4 (Bax)/B-cell lymphoma 2 (Bcl 2) ratio towards an anti-apoptotic constellation. Thus, PUFA n3 reveal a high neuroprotective and anti-inflammatory potential in an acute ischemic stroke model by targeting astroglial and microglial function as well as improving neuronal survival strategies. Our findings signify the potential clinical feasibility of PUFA n3 therapeutic treatment in stroke and other acute neurological diseases.

Sex Steroids Control Neuroinflammatory Processes in the Brain: Relevance for Acute Ischaemia and Degenerative Demyelination

Sex steroids have been demonstrated as powerful compounds to protect neurones and neural tissue from neurotoxic challenges and during neurodegeneration. A multitude of cellular actions have been attributed to female gonadal steroid hormones, including the regulation of pro‐survival and anti‐apoptotic factors, bioenergetic demands and radical elimination, growth factor allocation and counteracting against excitotoxicity. In recent years, immune‐modulatory and anti‐inflammatory characteristics of oestrogen and progesterone have also come under scrutiny. To date, each of these physiological responses has been considered to be partially and selectively integrated in the mediation of steroid‐mediated cell protection and tested in suitable animal models and in vitro systems. To what extent these individual effects contribute to the overall neural protection remains sketchy. One idea is that a battery of cellular mechanisms operates at the same time. On the other hand, interactions and the control of the brain‐intrinsic and peripheral immune system may play an additional and perhaps pioneering function in this scenario, notwithstanding the importance of secondary adjuvant mechanisms. In the present review, we highlight neuroprotective effects of oestrogen and progesterone in two different disease models of the brain, namely acute ischaemic and demyelination damage, which represent the most common acute and degenerative neurological disorders in humans. Besides other inflammatory parameters, we discuss the idea that chemokine expression and signalling appear to be early hallmarks in both diseases and are positively affected by sex steroids. In addition, the complex interplay with local brain‐resident immune‐competent cells appears to be controlled by the steroid environment.

Sex steroid hormone-mediated functional regulation of microglia-like BV-2 cells during hypoxia

17β-estradiol (E2) and progesterone (P) are neuroprotective hormones in different neurological disorders and in particular under hypoxic conditions in the brain. Both hormones dampen brain-intrinsic immune responses and regulate local glial cell function. Besides astrocytes which are functionally regulated in a manifold and complex manner, especially microglial cells are in the focus of steroid-mediated neuroprotection. In previous studies using a transient brain artery occlusion model, we demonstrated that microglial characteristics are critically modified after the administration of either E2 or P. We here studied the influence of sex steroids on the murine BV-2 microglia cell line under hypoxic conditions. Hypoxia changed the cell morphology from an amoeboid-like phenotype with processes to a rounded shape of secreting cell type. BV-2 cells expressed both estrogen receptor-β and progesterone receptors under each condition. Oxygen deprivation increased the expression of inducible nitric oxide synthetase (iNOS) and up-regulated selected cytokines and chemokines. Both hormones selectively prevented the induction of pro-inflammatory iNOS, interleukin IL-1ß, and chemokine ligand CCL5, whereas anti-inflammatory IL-10 and protective TREM 2 were up-regulated by sex steroids. Sex hormones abrogated hypoxia-dependent reduction of BV-2 phagocytic activity. We demonstrate that BV-2 microglia cells respond to hypoxia by enhanced pro-inflammatory cytokine secretion and reduced phagocytic activity. This effect is prevented by sex steroids resulting in a switch of BV-2 cells from a pro-inflammatory to a more anti-inflammatory phenotype. Anti-inflammatory effects of gonadal steroids might directly be mediated through hormone-microglia interactions in addition to known effects via astroglial regulation.

Hypoxia-induced gene expression of aquaporin-4, cyclooxygenase-2 and hypoxia-inducible factor 1α in rat cortical astroglia is inhibited by 17β-estradiol and progesterone.

17β-Estradiol (E2) and progesterone (P) are neuroprotective in acute brain injury by attenuating neuropathophysiological processes and regulating local glial function. Besides controlling brain-intrinsic immune responses, astrocytes are cellular targets for sex steroids in health and disease and typically resist to hypoxic damage. In this in vitro study, we aimed at uncovering astroglia-specific reactions to sublethal hypoxic conditions and astroglia-specific effects of both sex steroid hormones on these parameters. Short-term hypoxia for 3 h increased reactive oxygen species production, but had no influence on cell viability of cerebral cortical rat astroglia. Astrocytes expressed classical estrogen receptors (ER), progesterone receptor (PR), and a set of nonclassical steroid hormone receptors. Hypoxia specifically induced ERα and PR isoform A gene expression. Oxygen deprivation increased gene expression of aquaporin-4 (AQP4), hypoxia-inducible factor 1α (Hif1α), and cyclooxygenase-2 (COX2). The application of E2 and P selectively prevented this induction. Effects on protein levels of these genes appeared to be delayed. These data show that astrocytes change their receptivity for sex steroid hormones by switching steroid hormone receptor expression and that E2 and P modify or antagonize proinflammatory COX2 synthesis, edema-promoting AQP4 expression, and the Hif1α increase. In vivo studies have to address whether these cell responses contribute to steroid-mediated neuroprotection in stroke.