Microglia are continuously activated in the circumventricular organs of mouse brain.

Microglia are the primary resident immune cells of the brain parenchyma and transform into the amoeboid form in the "activated state" under pathological conditions from the ramified form in the "resting state" under physiologically healthy conditions. In the present study, we found that microglia in the circumventricular organs (CVOs) of adult mice displayed the amoeboid form with fewer branched cellular processes even under normal conditions; however, those in other brain regions showed the ramified form, which is characterized by well-branched and dendritic cellular processes. Moreover, microglia in the CVOs showed the strong protein expression of the M1 markers CD16/32 and CD86 and M2 markers CD206 and Ym1 without any pathological stimulation. Thus, the present results indicate that microglia in the CVOs of adult mice are morphologically and functionally activated under normal conditions, possibly due to the specialized features of the CVOs, namely, the entry of blood-derived molecules into parenchyma through fenestrated capillaries and the presence of neural stem cells.

Heterogeneous vascular permeability and alternative diffusion barrier in sensory circumventricular organs of adult mouse brain.

Fenestrated capillaries of the sensory circumventricular organs (CVOs), including the organum vasculosum of the lamina terminalis, the subfornical organ and the area postrema, lack completeness of the blood-brain barrier (BBB) to sense a variety of blood-derived molecules and to convey the information into other brain regions. We examine the vascular permeability of blood-derived molecules and the expression of tight-junction proteins in sensory CVOs. The present tracer assays revealed that blood-derived dextran 10 k (Dex10k) having a molecular weight (MW) of 10,000 remained in the perivascular space between the inner and outer basement membranes, but fluorescein isothiocyanate (FITC; MW: 389) and Dex3k (MW: 3000) diffused into the parenchyma. The vascular permeability of FITC was higher at central subdivisions than at distal subdivisions. Neither FITC nor Dex3k diffused beyond the dense network of glial fibrillar acidic protein (GFAP)-positive astrocytes/tanycytes. The expression of tight-junction proteins such as occludin, claudin-5 and zonula occludens-1 (ZO-1) was undetectable at the central subdivisions of the sensory CVOs but some was expressed at the distal subdivisions. Electron microscopic observation showed that capillaries were surrounded with numerous layers of astrocyte processes and dendrites. The expression of occludin and ZO-1 was also observed as puncta on GFAP-positive astrocytes/tanycytes of the sensory CVOs. Our study thus demonstrates the heterogeneity of vascular permeability and expression of tight-junction proteins and indicates that the outer basement membrane and dense astrocyte/tanycyte connection are possible alternative mechanisms for a diffusion barrier of blood-derived molecules, instead of the BBB.

Vascular endothelial growth factor-dependent angiogenesis and dynamic vascular plasticity in the sensory circumventricular organs of adult mouse brain.

The sensory circumventricular organs (CVOs), which comprise the organum vasculosum of the lamina terminalis (OVLT), the subfornical organ (SFO) and the area postrema (AP), lack a typical blood-brain barrier (BBB) and monitor directly blood-derived information to regulate body fluid homeostasis, inflammation, feeding and vomiting. Until now, almost nothing has been documented about vascular features of the sensory CVOs except fenestration of vascular endothelial cells. We therefore examine whether continuous angiogenesis occurs in the sensory CVOs of adult mouse. The angiogenesis-inducing factor vascular endothelial growth factor-A (VEGF-A) and the VEGF-A-regulating transcription factor hypoxia-inducible factor-1α were highly expressed in neurons of the OVLT and SFO and in both neurons and astrocytes of the AP. Expression of the pericyte-regulating factor platelet-derived growth factor B was high in astrocytes of the sensory CVOs. Immunohistochemistry of bromodeoxyuridine and Ki-67, a nuclear protein that is associated with cellular proliferation, revealed active proliferation of endothelial cells. Moreover, immunohistochemistry of caspase-3 and the basement membrane marker laminin showed the presence of apoptosis and sprouting of endothelial cells, respectively. Treatment with the VEGF receptor-associated tyrosine kinase inhibitor AZD2171 significantly reduced proliferation and filopodia sprouting of endothelial cells, as well as the area and diameter of microvessels. The mitotic inhibitor cytosine-b-D-arabinofuranoside reduced proliferation of endothelial cells and the vascular permeability of blood-derived low-molecular-weight molecules without changing vascular area and microvessel diameter. Thus, our data indicate that continuous angiogenesis is dependent on VEGF signaling and responsible for the dynamic plasticity of vascular structure and permeability.

Immunohistochemical detectability of cerebrovascular utrophin depends on the condition of basal lamina.

Utrophin is an autosomal homologue of dystrophin. Dystrophin is a member of the dystrophin-glycoprotein complex, which is a cell surface receptor for basal lamina components. In recent opinions utrophin occurs in the cerebrovascular endothelium but not in the perivascular glia. Cerebrovascular laminin immunoreactivity can only be detected in the subpial segments of the vessels, in circumventricular organs lacking blood-brain barrier, in immature vessels and following brain lesions. In our former experience utrophin immunoreactivity showed similar phenomena to that of laminin. The present study investigates the parallel occurrence of vascular utrophin and laminin immunoreactivity in the brain tissue, especially in the circumventricular organs, and during the parallel postnatal regression of both utrophin and laminin immunoreactivity. Their cerebrovascular immunoreactivity observed in frozen sections renders plausible the role of hidden but explorable epitopes, instead of a real absence of laminin and utrophin. The laminin epitopes are supposed to be hidden due to the fusion of the glial (i.e. brain parenchymal) and vascular basal laminae (Krum et al., Exp. Neurol. 111 (1991) 151). In all cases including its post-lesion re-appearance published formerly by us, laminin immunoreactivity may be attributed to the separation of glial and vascular basal laminae. Utrophin is localized, however, intracellularly, therefore a more complex molecular mechanism is to be assumed and it remains to be investigated how structural changes of the basal lamina may indirectly affect the immunoreactivity of utrophin. The results indicate that immunoreactivity may be influenced not only by the presence or absence of macromolecules but also by their functional state.