Blood–brain barrier genetic disruption leads to protective barrier formation at the Glia Limitans

Inflammation of the central nervous system (CNS) induces endothelial blood–brain barrier (BBB) opening as well as the formation of a tight junction barrier between reactive astrocytes at the Glia Limitans. We hypothesized that the CNS parenchyma may acquire protection from the reactive astrocytic Glia Limitans not only during neuroinflammation but also when BBB integrity is compromised in the resting state. Previous studies found that astrocyte-derived Sonic hedgehog (SHH) stabilizes the BBB during CNS inflammatory disease, while endothelial-derived desert hedgehog (DHH) is expressed at the BBB under resting conditions. Here, we investigated the effects of endothelial Dhh on the integrity of the BBB and Glia Limitans. We first characterized DHH expression within endothelial cells at the BBB, then demonstrated that DHH is down-regulated during experimental autoimmune encephalomyelitis (EAE). Using a mouse model in which endothelial Dhh is inducibly deleted, we found that endothelial Dhh both opens the BBB via the modulation of forkhead box O1 (FoxO1) transcriptional activity and induces a tight junctional barrier at the Glia Limitans. We confirmed the relevance of this glial barrier system in human multiple sclerosis active lesions. These results provide evidence for the novel concept of “chronic neuroinflammatory tolerance” in which BBB opening in the resting state is sufficient to stimulate a protective barrier at the Glia Limitans that limits the severity of subsequent neuroinflammatory disease. In summary, genetic disruption of the BBB generates endothelial signals that drive the formation under resting conditions of a secondary barrier at the Glia Limitans with protective effects against subsequent CNS inflammation. The concept of a reciprocally regulated CNS double barrier system has implications for treatment strategies in both the acute and chronic phases of multiple sclerosis pathophysiology.

Genetic disruption of the Blood Brain Barrier leads to protective barrier formation at the Glia Limitans

Recent work demonstrated that Central Nervous System (CNS) inflammation induces endothelial Blood Brain Barrier (BBB) opening as well as the formation of a tight junction barrier between reactive astrocytes at the Glia Limitans. We hypothesized that these two barriers may be reciprocally regulated by each other state and further, that the CNS parenchyma may acquire protection from the reactive astrocytic Glia Limitans not only in neuro-inflammation but also when BBB integrity is compromised under resting condition, without pathology. Previous studies identified Sonic hedgehog (Shh) astrocytic secretion as implicated in stabilizing the BBB during neuropathology and we recently demonstrated that desert hedgehog (Dhh) is expressed at the BBB in adults.Here we unraveled the role of the morphogen Dhh in maintaining BBB tightness and, using endothelial Dhh knockdown as a model of permeable BBB, we demonstrated that a double barrier system comprising both the BBB and Glia Limitans, is implemented in the CNS and regulated by a crosstalk going from endothelial cell to astrocytes.First, we showed that, under neuro-inflammatory conditions, Dhh expression is severely down regulated at the BBB and that Dhh is necessary for endothelial intercellular junction integrity as Dhh knockdown leads to CNS vascular leakage. We then demonstrated that, in Dhh endothelial knockout (DhhECKO) mice which display an open BBB, astrocytes are reactive and express the tight junction Claudin 4 (Cldn4) and showed that astrocytes can respond to signals secreted by the permeable endothelial BBB by becoming reactive and expressing Cldn4. To examine the consequences of the above results on disease severity, we finally induced multiple sclerosis in DhhECKO mice versus control littermates and showed that the pathology is less severe in the knockout animals due to Glia Limitans tightening, in response to BBB leakage, which drives inflammatory infiltrate entrapment into the perivascular space. Altogether these results suggest that genetic disruption of the BBB generates endothelial signals capable of driving the implementation of a secondary barrier at the Glia Limitans to protect the parenchyma. The concept of a reciprocally regulated CNS double barrier system has implications for treatment strategies in both the acute and chronic phases of multiple sclerosis pathophysiology.

Structure and Junctional Complexes of Endothelial, Epithelial and Glial Brain Barriers

The homeostasis of the central nervous system (CNS) is ensured by the endothelial, epithelial, mesothelial and glial brain barriers, which strictly control the passage of molecules, solutes and immune cells. While the endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid barrier (BCSFB) have been extensively investigated, less is known about the epithelial and mesothelial arachnoid barrier and the glia limitans. Here, we summarize current knowledge of the cellular composition of the brain barriers with a specific focus on describing the molecular constituents of their junctional complexes. We propose that the brain barriers maintain CNS immune privilege by dividing the CNS into compartments that differ with regard to their role in immune surveillance of the CNS. We close by providing a brief overview on experimental tools allowing for reliable in vivo visualization of the brain barriers and their junctional complexes and thus the respective CNS compartments.

Angiocentric astrocytoma in a cat

Gliomas are common primary central nervous system neoplasms of dogs and cats, but atypical glioma subtypes are rare. Herein we report an angiocentric astrocytoma in a 15-y-old spayed female domestic shorthaired cat that was euthanized after therapy-resistant seizures. Gross anatomic changes consisted of swelling of the rostral leptomeninges over the olfactory bulbs and rostral telencephalon. Histologically, polygonal-to-elongate atypical neoplastic cells were arranged along perivascular spaces within these areas. Neoplastic cells were positive for glial fibrillary acidic protein, S100 protein, and vimentin. Ultrastructurally, round-to-elongate neoplastic cells emitting long processes with aggregates of intermediary filaments expanded and occupied the spaces between the vascular basement membrane and the glia limitans; nuclei had marginal and central heterochromatin. Tight junctions connected the plasma membrane of neighboring cells. The cell morphology, immunohistochemistry, and ultrastructural findings were consistent with an astrocytoma; the exclusive perivascular arrangement of neoplastic cells with no parenchymal mass warranted the diagnosis of angiocentric astrocytoma.

Iba1+ Microglia Exhibit Morphological Differences between Inferior Colliculus Sub-Regions and Their Abutments onto GAD67+ Somata Reveal Two Novel Sub-types of GABAergic Neuron

Microglia have classically been viewed as the endogenous phagocytes of the brain, however, emerging evidence suggests roles for microglia in the healthy, mature nervous system. We know little of the contribution microglia make to ongoing processing in sensory systems. To explore Iba1+ microglial diversity, we employed the inferior colliculi (IC) as model nuclei, as they are characterized by sub-regions specialized for differing aspects of auditory processing. We conducted fluorescent multi-channel immunohistochemistry and confocal microscopy in guinea pigs of both sexes and discovered that the density and morphology of Iba1+ labelling varied between parenchymal sub-regions of IC, while GFAP+ labelling of astrocytes was confined to the glia limitans externa and peri-vascular regions. The density of Iba1+ microglia somata was similar across sub-regions, however a greater amount of labelling was found in dorsal cortex than ventral central nucleus or lateral cortex. To further understand these differences between sub-regions in IC, Sholl and skeleton analyses of individual microglia revealed a greater number of branching ramifications in dorsal cortex. We also quantified abutments of Iba1+ microglial processes onto GAD67+ (putative GABAergic) somata. Cluster analyses revealed two novel sub-types of GAD67+ neuron, which can be distinguished solely based on the quantity of axo-somatic Iba1+ abutments they receive. These data demonstrate Iba1+ microglia exhibit different morphologies and interactions with GAD67+ neurons in distinct sub-regions of the mature, healthy IC. Taken together, these findings suggest significant heterogeneity amongst microglia in the auditory system, possibly related to the ongoing functional demands of their niche.