Analysis of rodent brains with X-ray fluorescence (XRF) microscopy combined with immunohistochemistry allowed us to demonstrate that local Cu concentrations are thousands of times higher in the glia of the subventricular zone than in other cells. Using XRF microscopy with subcellular resolution and intracellular X-ray absorption spectroscopy we determined the copper (I) oxidation state and the sulfur ligand environment. Cu K-edge XANES is consistent with Cu being bound as a multimetallic Cu-S cluster similar to one present in Cu-metallothionein. Analysis of age related changes show that Cu content in astrocytes of the SVZ increases 4 fold from 3 weeks to 9 months while Cu concentration in other brain areas remain essentially constant. This increase in Cu correlates with a decrease in adult neurogenesis assessed using the Ki67 marker (both, however, can be age related effects). We demonstrate that the Cu distribution and age-related concentration changes in the brain are highly cell-specific.

Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently described a model combining noninflammatory cytodegeneration (via cuprizone) with the classic active experimental autoimmune encephalomyelitis (Cup/EAE model), which exhibits inflammatory forebrain lesions. Here, we describe the histopathological characteristics and progression of these Cup/EAE lesions. We show that inflammatory lesions develop at various topographical sites in the forebrain, including white matter tracts and cortical and subcortical grey matter areas. The lesions are characterized by focal demyelination, discontinuation of the perivascular glia limitans, focal axonal damage, and neutrophil granulocyte extravasation. Transgenic mice with enhanced green fluorescent protein-expressing microglia and red fluorescent protein-expressing monocytes reveal that both myeloid cell populations contribute to forebrain inflammatory infiltrates. EAE-triggered inflammatory cerebellar lesions were augmented in mice pre-intoxicated with cuprizone. Gene expression studies suggest roles of the chemokines Cxcl10, Ccl2, and Ccl3 in inflammatory lesion formation. Finally, follow-up experiments in Cup/EAE mice with chronic disease revealed that forebrain, but not spinal cord, lesions undergo spontaneous reorganization and repair. This study underpins the significance of brain-intrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.

The expression of the endothelial and inducible nitric oxide synthases (eNOS and iNOS, respectively) was examined in the endometrium of cyclic and pregnant mares by real-time polymerase chain reaction and immunohistology. The concentration of eNOS mRNA varied throughout the oestrous cycle, with significantly higher transcripts on Day 5 of the oestrous cycle (P < 0.05), whereas iNOS transcription did not change significantly over time (P > 0.05). In early pregnant mares both eNOS and iNOS mRNA increased between Days 12 and 15 (P < 0.05). In cyclic mares, eNOS protein was detected immunocytochemically in endometrial epithelia, the basement membrane, the endothelial layer and smooth muscle cells of the vasculature. Using immunocytochemical methods, iNOS protein was undetectable in the endometrium of cyclic mares but could be demonstrated in pregnant mares. Endometrial epithelia of pregnant mares were immunopositive for both proteins with a more intense labelling for iNOS. Thus, the present study describes for the first time the modulation and spatial distribution of eNOS and iNOS expression during the oestrous cycle and early pregnancy, suggesting that ovarian steroids are differently involved in the regulation of each NOS. Localisation of eNOS protein in endometrial epithelia and various vascular components indicates that this isoform may be involved in the regulation of endometrial cyclicity. The presence and increase of both forms of NOS during early gestation suggest a role for them in the control of endometrial vascular bed and glandular activity to provide a suitable microenvironment for successful pregnancy.

Brain-intrinsic degenerative cascades are a proposed factor driving inflammatory lesion formation in multiple sclerosis (MS) patients. We recently showed that encephalitogenic lymphocytes are recruited to the sites of active demyelination induced by cuprizone. Here, we investigated whether cuprizoneinduced oligodendrocyte and myelin pathology is sufficient to trigger peripheral immune cell recruitment into the forebrain. We show that early cuprizone-induced white matter lesions display a striking similarity to early MS lesions, i.e., oligodendrocyte degeneration, microglia activation and absence of severe lymphocyte infiltration. Such early cuprizone lesions are sufficient to trigger peripheral immune cell recruitment secondary to subsequent EAE (experimental autoimmune encephalomyelitis) induction. The lesions are characterized by discontinuation of the perivascular glia limitans, focal axonal damage, and perivascular astrocyte pathology. Time course studies showed that the severity of cuprizone-induced lesions positively correlates with the extent of peripheral immune cell recruitment. Furthermore, results of genome-wide array analyses suggest that moesin is integral for early microglia activation in cuprizone and MS lesions. This study underpins the significance of brainintrinsic degenerative cascades for immune cell recruitment and, in consequence, MS lesion formation.

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.