The choroid plexus maintains adult brain ventricles and subventricular zone neuroblast pool, which facilitates poststroke neurogenesis.

The brain's neuroreparative capacity after injuries such as ischemic stroke is partly contained in the brain's neurogenic niches, primarily the subventricular zone (SVZ), which lies in close contact with the cerebrospinal fluid (CSF) produced by the choroid plexus (ChP). Despite the wide range of their proposed functions, the ChP/CSF remain among the most understudied compartments of the central nervous system (CNS). Here, we report a mouse genetic tool (the ROSA26iDTR mouse line) for noninvasive, specific, and temporally controllable ablation of CSF-producing ChP epithelial cells to assess the roles of the ChP and CSF in brain homeostasis and injury. Using this model, we demonstrate that ChP ablation causes rapid and permanent CSF volume loss in both aged and young adult brains, accompanied by disruption of ependymal cilia bundles. Surprisingly, ChP ablation did not result in overt neurological deficits at 1 mo postablation. However, we observed a pronounced decrease in the pool of SVZ neuroblasts (NBs) following ChP ablation, which occurs due to their enhanced migration into the olfactory bulb. In the middle cerebral artery occlusion model of ischemic stroke, NB migration into the lesion site was also reduced in the CSF-depleted mice. Thus, our study establishes an important role of ChP/CSF in regulating the regenerative capacity of the adult brain under normal conditions and after ischemic stroke.

Preferential delivery of lipid-ligand conjugated DNA/RNA heteroduplex oligonucleotide to ischemic brain in hyperacute stage.

Antisense oligonucleotide (ASO) is a major tool used for silencing pathogenic genes. For stroke in the hyperacute stage, however, the ability of ASO to regulate genes is limited by its poor delivery to the ischemic brain owing to sudden occlusion of the supplying artery. Here we show that, in a mouse model of permanent ischemic stroke, lipid-ligand conjugated DNA/RNA heteroduplex oligonucleotide (lipid-HDO) was unexpectedly delivered 9.6 times more efficiently to the ischemic area of the brain than to the contralateral non-ischemic brain and achieved robust gene knockdown and change of stroke phenotype, despite a 90% decrease in cerebral blood flow in the 3 h after occlusion. This delivery to neurons was mediated via receptor-mediated transcytosis by lipoprotein receptors in brain endothelial cells, the expression of which was significantly upregulated after ischemia. This study provides proof-of-concept that lipid-HDO is a promising gene-silencing technology for stroke treatment in the hyperacute stage.

The Anti-Inflammatory Agent Bindarit Attenuates the Impairment of Neural Development through Suppression of Microglial Activation in a Neonatal Hydrocephalus Mouse Model.

Neonatal hydrocephalus presents with various degrees of neuroinflammation and long-term neurologic deficits in surgically treated patients, provoking a need for additional medical treatment. We previously reported elevated neuroinflammation and severe periventricular white matter damage in the progressive hydrocephalus (prh) mutant which contains a point mutation in the Ccdc39 gene, causing loss of cilia-mediated unidirectional CSF flow. In this study, we identified cortical neuropil maturation defects such as impaired excitatory synapse maturation and loss of homeostatic microglia, and swimming locomotor defects in early postnatal prh mutant mice. Strikingly, systemic application of the anti-inflammatory small molecule bindarit significantly supports healthy postnatal cerebral cortical development in the prh mutant. While bindarit only mildly reduced the ventricular volume, it significantly improved the edematous appearance and myelination of the corpus callosum. Moreover, the treatment attenuated thinning in cortical Layers II-IV, excitatory synapse formation, and interneuron morphogenesis, by supporting the ramified-shaped homeostatic microglia from excessive cell death. Also, the therapeutic effect led to the alleviation of a spastic locomotor phenotype of the mutant. We found that microglia, but not peripheral monocytes, contribute to amoeboid-shaped activated myeloid cells in prh mutants' corpus callosum and the proinflammatory cytokines expression. Bindarit blocks nuclear factor (NF)-kB activation and its downstream proinflammatory cytokines, including monocyte chemoattractant protein-1, in the prh mutant. Collectively, we revealed that amelioration of neuroinflammation is crucial for white matter and neuronal maturation in neonatal hydrocephalus. Future studies of bindarit treatment combined with CSF diversion surgery may provide long-term benefits supporting neuronal development in neonatal hydrocephalus.SIGNIFICANCE STATEMENT In neonatal hydrocephalus, little is known about the signaling cascades of neuroinflammation or the impact of such inflammatory insults on neural cell development within the perinatal cerebral cortex. Here, we report that proinflammatory activation of myeloid cells, the majority of which are derived from microglia, impairs periventricular myelination and cortical neuronal maturation using the mouse prh genetic model of neonatal hydrocephalus. Administration of bindarit, an anti-inflammatory small molecule that blocks nuclear factor (NF)-kB activation, restored the cortical thinning and synaptic maturation defects in the prh mutant brain through suppression of microglial activation. These data indicate the potential therapeutic use of anti-inflammatory reagents targeting neuroinflammation in the treatment of neonatal hydrocephalus.

Sphingosine-1-Phosphate Receptor 1 Activation Enhances Leptomeningeal Collateral Development and Improves Outcome after Stroke in Mice.

BACKGROUND: Development of collateral circulation after acute ischemic stroke is triggered by shear stress that occurs in pre-existing arterioles. Recently, sphingosine-1-phosphate receptor 1 (S1P1) on endothelial cells was reported to sense shear stress and transduce its signaling pathways. METHODS: BALB/c mice (n = 118) were subjected to permanent middle cerebral artery occlusion (pMCAO) or sham operation. We investigated the effect of an S1P1-selective agonist SEW2871 on leptomeningeal collateral arteries and neurological outcome after pMCAO. RESULTS: Immunohistochemistry showed that without treatment, the expression of S1P1 on endothelial cells of leptomeningeal arteries and capillaries increased early after pMCAO, peaking at 6 hours, whereas a significant increase in the expression of S1P1 in neurons was seen from 24 hours later. After intraperitoneal administration of SEW2871 for 7 days after pMCAO, the number of leptomeningeal collateral arteries was significantly increased, cerebral blood flow improved, infarct volume was decreased, and neurological outcome improved compared with the controls. Significantly increased phosphorylation of endothelial nitric oxide synthase (eNOS) as early as 6 hours after pMCAO and higher expression of tight junction proteins at postoperative day 3 were observed with SEW2871 treatment as assessed by Western blot. Daily administration of SEW2871 also increased capillary density in peri-infarct regions and promoted monocyte/macrophage mobilization to the surface of ischemic cortex at 7 days after pMCAO. CONCLUSIONS: An S1P1-selective agonist enhanced leptomeningeal collateral circulation via eNOS phosphorylation and promoted postischemic angiogenesis with reinforced blood-brain barrier integrity in a mouse model of acute ischemic stroke, leading to smaller infarct volume and better neurological outcome.

The choroid plexus maintains ventricle volume and adult subventricular zone neuroblast pool, which facilitates post-stroke neurogenesis.

The brain's neuroreparative capacity after injuries such as ischemic stroke is contained in the brain's neurogenic niches, primarily the subventricular zone (SVZ), which lies in close contact with the cerebrospinal fluid (CSF) produced by the choroid plexus (ChP). Despite the wide range of their proposed functions, the ChP/CSF remain among the most understudied compartments of the central nervous system (CNS). Here we report a mouse genetic tool (the ROSA26iDTR mouse line) for non-invasive, specific, and temporally controllable ablation of CSF-producing ChP epithelial cells to assess the roles of the ChP and CSF in brain homeostasis and injury. Using this model, we demonstrate that ChP ablation causes rapid and permanent CSF volume loss accompanied by disruption of ependymal cilia bundles. Surprisingly, ChP ablation did not result in overt neurological deficits at one-month post-ablation. However, we observed a pronounced decrease in the pool of SVZ neuroblasts following ChP ablation, which occurs due to their enhanced migration into the olfactory bulb. In the MCAo model of ischemic stroke, neuroblast migration into the lesion site was also reduced in the CSF-depleted mice. Thus, our study establishes an important and novel role of ChP/CSF in regulating the regenerative capacity of the adult brain under normal conditions and after ischemic stroke.

Early postnatal microglial ablation in the Ccdc39 mouse model reveals adverse effects on brain development and in neonatal hydrocephalus.

BACKGROUND: Neonatal hydrocephalus is a congenital abnormality resulting in an inflammatory response and microglial cell activation both clinically and in animal models. Previously, we reported a mutation in a motile cilia gene, Ccdc39 that develops neonatal progressive hydrocephalus (prh) with inflammatory microglia. We discovered significantly increased amoeboid-shaped activated microglia in periventricular white matter edema, reduced mature homeostatic microglia in grey matter, and reduced myelination in the prh model. Recently, the role of microglia in animal models of adult brain disorders was examined using cell type-specific ablation by colony-stimulating factor-1 receptor (CSF1R) inhibitor, however, little information exists regarding the role of microglia in neonatal brain disorders such as hydrocephalus. Therefore, we aim to see if ablating pro-inflammatory microglia, and thus suppressing the inflammatory response, in a neonatal hydrocephalic mouse line could have beneficial effects. METHODS: In this study, Plexxikon 5622 (PLX5622), a CSF1R inhibitor, was subcutaneously administered to wild-type (WT) and prh mutant mice daily from postnatal day (P) 3 to P7. MRI-estimated brain volume was compared with untreated WT and prh mutants P7-9 and immunohistochemistry of the brain sections was performed at P8 and P18-21. RESULTS: PLX5622 injections successfully ablated IBA1-positive microglia in both the WT and prh mutants at P8. Of the microglia that are resistant to PLX5622 treatment, there was a higher percentage of amoeboid-shaped microglia, identified by morphology with retracted processes. In PLX-treated prh mutants, there was increased ventriculomegaly and no change in the total brain volume was observed. Also, the PLX5622 treatment significantly reduced myelination in WT mice at P8, although this was recovered after full microglia repopulation by P20. Microglia repopulation in the mutants worsened hypomyelination at P20. CONCLUSIONS: Microglia ablation in the neonatal hydrocephalic brain does not improve white matter edema, and actually worsens ventricular enlargement and hypomyelination, suggesting critical functions of homeostatic ramified microglia to better improve brain development with neonatal hydrocephalus. Future studies with detailed examination of microglial development and status may provide a clarification of the need for microglia in neonatal brain development.

Effective silencing of miR-126 after ischemic stroke by means of intravenous α-tocopherol-conjugated heteroduplex oligonucleotide in mice.

Brain endothelial cells (BECs) are involved in the pathogenesis of ischemic stroke. Recently, several microRNAs (miRNAs) in BECs were reported to regulate the endothelial function in ischemic brain. Therefore, modulation of miRNAs in BECs by a therapeutic oligonucleotide to inhibit miRNA (antimiR) could be a useful strategy for treating ischemic stroke. However, few attempts have been made to achieve this strategy via systemic route due to lack of efficient delivery-method toward BECs. Here, we have developed a new technology for delivering an antimiR into BECs and silencing miRNAs in BECs, using a mouse ischemic stroke model. We designed a heteroduplex oligonucleotide, comprising an antimiR against miRNA-126 (miR-126) known as the endothelial-specific miRNA and its complementary RNA, conjugated to α-tocopherol as a delivery ligand (Toc-HDO targeting miR-126). Intravenous administration of Toc-HDO targeting miR-126 remarkably suppressed miR-126 expression in ischemic brain of the model mice. In addition, we showed that Toc-HDO targeting miR-126 was delivered into BECs more efficiently than the parent antimiR in ischemic brain, and that it was delivered more effectively in ischemic brain than non-ischemic brain of this model mice. Our study highlights the potential of this technology as a new clinical therapeutic option for ischemic stroke.

Impaired neural differentiation and glymphatic CSF flow in the Ccdc39 rat model of neonatal hydrocephalus: genetic interaction with L1cam.

Neonatal hydrocephalus affects about one child per 1000 births and is a major congenital brain abnormality. We previously discovered a gene mutation within the coiled-coil domain-containing 39 (Ccdc39) gene, which causes the progressive hydrocephalus (prh) phenotype in mice due to lack of ependymal-cilia-mediated cerebrospinal fluid (CSF) flow. In this study, we used CRISPR/Cas9 to introduce the Ccdc39 gene mutation into rats, which are more suitable for imaging and surgical experiments. The Ccdc39prh/prh mutants exhibited mild ventriculomegaly at postnatal day (P)5 that progressed into severe hydrocephalus by P11 (P<0.001). After P11, macrophage and neutrophil invasion along with subarachnoid hemorrhage were observed in mutant brains showing reduced neurofilament density, hypomyelination and increased cell death signals compared with wild-type brains. Significantly more macrophages entered the brain parenchyma at P5 before hemorrhaging was noted and increased expression of a pro-inflammatory factor (monocyte chemoattractant protein-1) was found in the cortical neural and endothelial cells in the mutant brains at P11. Glymphatic-mediated CSF circulation was progressively impaired along the middle cerebral artery from P11 as mutants developed severe hydrocephalus (P<0.001). In addition, Ccdc39prh/prh mutants with L1 cell adhesion molecule (L1cam) gene mutation, which causes X-linked human congenital hydrocephalus, showed an accelerated early hydrocephalus phenotype (P<0.05-0.01). Our findings in Ccdc39prh/prh mutant rats demonstrate a possible causal role of neuroinflammation in neonatal hydrocephalus development, which involves impaired cortical development and glymphatic CSF flow. Improved understanding of inflammatory responses and the glymphatic system in neonatal hydrocephalus could lead to new therapeutic strategies for this condition.This article has an associated First Person interview with the joint first authors of the paper.

Long-term Effects of Intravenous Cyclophosphamide in Combination with Mesna Provided Intravenously and via Bladder Perfusion in a Patient with Severe Multifocal Motor Neuropathy.

A 25-year-old woman presenting with progressive muscle weakness in the distal extremities in the absence of sensory involvement for 2 years was diagnosed with multifocal motor neuropathy (MMN). Her disease was difficult to manage with various immunosuppressants, and the muscle weakness eventually progressed to involve the respiratory muscles, necessitating mechanical ventilation. Intravenous cyclophosphamide (CY) dramatically improved her symptoms, and she has since maintained her ambulatory status for 18 years with intermittent CY therapy. Because the patient presented with hemorrhagic cystitis due to CY, we also implemented mesna administration by bladder perfusion. The administration of CY should therefore be considered in patients with severe MMN that is unresponsive to standard therapy.

Acute development of collateral circulation and therapeutic prospects in ischemic stroke.

In acute ischemic stroke, collateral circulation plays an important role in maintaining blood flow to the tissue that is at risk of progressing into ischemia, and in increasing the successful recanalization rate without hemorrhagic transformation. We have reported that well-developed collateral circulation is associated with smaller infarct volume and better long-term neurological outcome, and it disappears promptly once the effective recanalization is achieved. Contrary to the belief that collateral vessels develop over time in chronic stenotic condition, there exists a phenomenon that collateral circulation develops immediately in acute stenosis or occlusion of the arteries and it seems to be triggered by fluid shear stress, which occurs between the territories of stenotic/occluded arteries and those fed by surrounding intact arteries. We believe that this acute development of collateral circulation is a target of novel therapeutics in ischemic stroke and refer our recent attempt in enhancing collateral circulation by modulating sphingosine-1-phosphate receptor 1, which is a known shear-stress mechanosensing protein.

A patient with infective endocarditis caused by community-acquired Pseudomonas aeruginosa infection.

An 85-year-old woman complaining of nausea was admitted to our hospital after being found to have complete atrioventricular block. We diagnosed the patient with infective endocarditis after observing vegetation on transesophageal echocardiography (TEE) and detecting Pseudomonas aeruginosa in a blood culture. The patient had no history of intravenous drug use, instrumentation or valvular disease. Although sensitive antibiotics were administered intravenously, the second TEE performed on the 10th day demonstrated increased vegetation. The patient developed septic shock and died on the 14th day. To our knowledge, this is the first report of infective endocarditis caused by community-acquired Pseudomonas aeruginosa in Japan.