The Cuprizone Mouse Model: A Comparative Study of Cuprizone Formulations from Different Manufacturers.

The Cuprizone mouse model is widely used in studies on de- and remyelination. In the hands of different experimenters, the Cuprizone concentrations that lead to comparable levels of demyelination differ considerably. The reasons for this variability are unknown. In this study, we tested whether different Cuprizone formulations from different vendors and manufacturers influenced Cuprizone-induced histopathological hallmarks. We intoxicated male C57BL/6 mice with six Cuprizone powders that differed in their manufacturer, vendor, and purity. After five weeks, we analyzed the body weight changes over the course of the experiment, as well as the demyelination, astrogliosis, microgliosis and axonal damage by histological LFB-PAS staining and immunohistochemical labelling of PLP, IBA1, GFAP and APP. All Cuprizone formulations induced demyelination, astrogliosis, microgliosis, axonal damage and a moderate drop in body weight at the beginning of the intoxication period. In a cumulative evaluation of all analyses, two Cuprizone formulations performed weaker than the other formulations. In conclusion, all tested formulations did work, but the choice of Cuprizone formulation may have been responsible for the considerable variability in the experimental outcomes.

Morphology of the murine choroid plexus: Attachment regions and spatial relation to the subarachnoid space.

The choroid plexus has recently been identified as a possible migration route for peripheral immune cells into the central nervous system. For future investigation of this route, profound knowledge of the morphology of the murine choroid plexus is a prerequisite. We here present a detailed morphological description of the murine choroid plexus, its attachment regions as well as its spatial relation to the subarachnoid space. We used micro-computed tomography of immersion-contrasted fixated brains to generate three-dimensional models of the ventricle system and the choroid plexus and aligned micro-computed tomography-based sections with histological paraffin-embedded sections after immunohistochemical labeling of the basal lamina and choroid plexus epithelium marker proteins (laminin and aquaporin 1). The murine choroid plexus is located in all four ventricles and is attached to the brain parenchyma in narrow attachment regions with a specific morphology in each ventricle. While in the lateral and fourth ventricle, the attachment site is formed by thin tissue bridges, the choroid plexus attachment in the third ventricle has a more complex V-like shape. In all ventricles, the choroid plexus is in close spatial relationship with the subarachnoid space that extends from the brain surface along physiologic openings toward the choroid plexus. In summary, we here provide a description of the morphology of the murine ventricle system and choroid plexus, the attachment regions of the choroid plexus and its connection to the subarachnoid space, as well as a three-dimensional model of the ventricles, the choroid plexus, and the subarachnoid space to facilitate a spatial understanding of these complex structures.

Transmembrane protein 119 is neither a specific nor a reliable marker for microglia.

Microglia are the resident innate immune cells of the central nervous system (CNS) parenchyma. To determine the impact of microglia on disease development and progression in neurodegenerative and neuroinflammatory diseases, it is essential to distinguish microglia from peripheral macrophages/monocytes, which are eventually equally recruited. It has been suggested that transmembrane protein 119 (TMEM119) serves as a reliable microglia marker that discriminates resident microglia from blood-derived macrophages in the human and murine brain. Here, we investigated the validity of TMEM119 as a microglia marker in four in vivo models (cuprizone intoxication, experimental autoimmune encephalomyelitis (EAE), permanent filament middle cerebral artery occlusion (fMCAo), and intracerebral 6-hydroxydopamine (6-OHDA) injections) as well as post mortem multiple sclerosis (MS) brain tissues. In all applied animal models and post mortem MS tissues, we found increased densities of ionized calcium-binding adapter molecule 1+ (IBA1+ ) cells, paralleled by a significant decrease in TMEM119 expression. In addition, other cell types in peripheral tissues (i.e., follicular dendritic cells and brown adipose tissue) were also found to express TMEM119. In summary, this study demonstrates that TMEM119 is not exclusively expressed by microglia nor does it label all microglia, especially under cellular stress conditions. Since novel transgenic lines have been developed to label microglia using the TMEM119 promotor, downregulation of TMEM119 expression might interfere with the results and should, thus, be considered when working with these transgenic mouse models.

What Guides Peripheral Immune Cells into the Central Nervous System?

Multiple sclerosis (MS), an immune-mediated demyelinating disease of the central nervous system (CNS), initially presents with a relapsing-remitting disease course. During this early stage of the disease, leukocytes cross the blood-brain barrier to drive the formation of focal demyelinating plaques. Disease-modifying agents that modulate or suppress the peripheral immune system provide a therapeutic benefit during relapsing-remitting MS (RRMS). The majority of individuals with RRMS ultimately enter a secondary progressive disease stage with a progressive accumulation of neurologic deficits. The cellular and molecular basis for this transition is unclear and the role of inflammation during the secondary progressive disease stage is a subject of intense and controversial debate. In this review article, we discuss the following main hypothesis: during both disease stages, peripheral immune cells are triggered by CNS-intrinsic stimuli to invade the brain parenchyma. Furthermore, we outline the different neuroanatomical routes by which peripheral immune cells might migrate from the periphery into the CNS.

Cuprizone-induced demyelination triggers a CD8-pronounced T cell recruitment.

The loss of myelinating oligodendrocytes is a key characteristic of many neurological diseases, including Multiple Sclerosis (MS). In progressive MS, where effective treatment options are limited, peripheral immune cells can be found at the site of demyelination and are suggested to play a functional role during disease progression. In this study, we hypothesize that metabolic oligodendrocyte injury, caused by feeding the copper chelator cuprizone, is a potent trigger for peripheral immune cell recruitment into the central nervous system (CNS). We used immunohistochemistry and flow cytometry to evaluate the composition, density, and activation status of infiltrating T lymphocytes in cuprizone-intoxicated mice and post-mortem progressive MS tissues. Our results demonstrate a predominance of CD8+ T cells along with high proliferation rates and cytotoxic granule expression, indicating an antigenic and pro-inflammatory milieu in the CNS of cuprizone-intoxicated mice. Numbers of recruited T cells and the composition of lymphocytic infiltrates in cuprizone-intoxicated mice were found to be comparable to those found in progressive MS lesions. Finally, amelioration of the cuprizone-induced pathology by treating mice with laquinimod significantly reduces the number of recruited T cells. Overall, this study provides strong evidence that toxic demyelination is a sufficient trigger for T cells to infiltrate the demyelinated CNS. Further investigation of the mode of action and functional consequence of T cell recruitment might offer promising new therapeutic approaches for progressive MS.

Aquaporin-4 Expression during Toxic and Autoimmune Demyelination.

The water channel protein aquaporin-4 (AQP4) is required for a normal rate of water exchange across the blood-brain interface. Following the discovery that AQP4 is a possible autoantigen in neuromyelitis optica, the function of AQP4 in health and disease has become a research focus. While several studies have addressed the expression and function of AQP4 during inflammatory demyelination, relatively little is known about its expression during non-autoimmune-mediated myelin damage. In this study, we used the toxin-induced demyelination model cuprizone as well as a combination of metabolic and autoimmune myelin injury (i.e., Cup/EAE) to investigate AQP4 pathology. We show that during toxin-induced demyelination, diffuse AQP4 expression increases, while polarized AQP4 expression at the astrocyte endfeet decreases. The diffuse increased expression of AQP4 was verified in chronic-active multiple sclerosis lesions. Around inflammatory brain lesions, AQP4 expression dramatically decreased, especially at sites where peripheral immune cells penetrate the brain parenchyma. Humoral immune responses appear not to be involved in this process since no anti-AQP4 antibodies were detected in the serum of the experimental mice. We provide strong evidence that the diffuse increase in anti-AQP4 staining intensity is due to a metabolic injury to the brain, whereas the focal, perivascular loss of anti-AQP4 immunoreactivity is mediated by peripheral immune cells.

High Speed Ventral Plane Videography as a Convenient Tool to Quantify Motor Deficits during Pre-Clinical Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is the most commonly used multiple sclerosis animal model. EAE mice typically develop motor deficits in a caudal-to-rostral pattern when inflammatory lesions have already developed. However, to monitor more subtle behavioral deficits during lesion development (i.e., pre-clinical phase), more sophisticated methods are needed. Here, we investigated whether high speed ventral plane videography can be applied to monitor early motor deficits during 'pre-clinical' EAE. For this purpose, EAE was induced in C57BL/6 mice and gait abnormalities were quantified using the DigiGait™ apparatus. Gait deficits were related to histopathological changes. 10 out of 10 control (100%), and 14 out of 18 (77.8%) pre-clinical EAE mice could be evaluated using DigiGait™. EAE severity was not influenced by DigiGait™-related mice handlings. Most gait parameters recorded from day 6 post-immunization until the end of the experiment were found to be stable in control mice. During the pre-clinical phase, when conventional EAE scorings failed to detect any functional impairment, EAE mice showed an increased Swing Time, increased %Swing Stride, decreased %Stance Stride, decreased Stance/Swing, and an increased Absolute Paw Angle. In summary, DigiGait™ is more sensitive than conventional scoring approaches to study motor deficits during the EAE pre-clinical phase.