MicroRNAs regulate neural stem cell function. Argonaute 2 protein, constituent of the RNA-induced silencing complex, plays an important role in regulating microRNA function for post-transcriptional gene silencing. Although Argonaute 2 and microRNAs are recognized as central regulators of RNA-induced silencing complex, their precise role in adult neural stem cell function has remained unclear. In particular, it was not known whether Argonaute 2 is required for sustaining neural stem cell proliferation, neurogenesis, and oligodendrogenesis in the adult brain, or how its loss might influence recovery after ischemic injury. The present study examined the effect of Argonaute 2 deletion in adult neural stem cells on neurogenesis and oligodendrogenesis. Adult transgenic mice with conditional and inducible ablation of Argonaute 2 in Ascl1-lineage neural stem cells exhibited the reduction of neurogenesis in the ventricular-subventricular zone of the lateral ventricle and in the subgranular zone of the dentate gyrus, as evidenced by a decrease in neural stem cell proliferation and neuroblast numbers. Argonaute 2 deletion also reduced oligodendrogenesis in the corpus callosum, as indicated by the reduction of oligodendrocyte progenitor cell proliferation and the number of mature oligodendrocytes. Additionally, deleting Argonaute 2 in adult neural stem cells of ischemic mice exacerbated impairments of sensorimotor and cognitive functions. Mechanistically, Argonaute 2 ablation in neural stem cells reduced the stability of mature microRNAs and downregulated genes involved in the Shh (Sonic Hedgehog), Notch, and TGF beta (transforming growth factor beta) signaling pathways, which regulate the functions of neural stem cells. Collectively, our study demonstrates that Argonaute 2 is essential for adult neural stem cell-mediated neurogenesis and oligodendrogenesis, with its deletion worsening recovery after ischemia. By revealing that Argonaute 2 stabilizes mature microRNAs, our work uncovers a novel mechanism of neural stem cell regulation and highlights Argonaute 2 as a potential therapeutic target for neurodegenerative and ischemic brain diseases.

The glymphatic system suggests the convective bulk flow of cerebrospinal fluid (CSF) through perivascular spaces and the interstitial spaces of the brain parenchyma for the rapid removal of toxic waste solutes from the brain. However, the presence of convective bulk flow within the brain interstitial spaces is still under debate. We first addressed this argument to determine the involvement of the glymphatic system in brain waste clearance utilizing contrast-enhanced 3D T1-weighted imaging (T1WI), diffusion tensor imaging (DTI), and confocal microscopy imaging. Furthermore, perivascular macrophages (PVMs), which are immune cells located within perivascular spaces, have not been thoroughly explored for their association with the glymphatic system. Therefore, we investigated tracer uptake by PVMs in the perivascular spaces of both the arteries/arterioles and veins/venules and the potential association of PVMs in assisting the glymphatic system for interstitial waste clearance. Our findings demonstrated that both convective bulk flow and diffusion are responsible for the clearance of interstitial waste solutes from the brain parenchyma. Furthermore, our results suggested that PVMs may play an important function in glymphatic system-mediated interstitial waste clearance. The glymphatic system and PVMs could be targeted to enhance interstitial waste clearance in patients with waste-associated neurological conditions and aging.

Cerebrospinal fluid (CSF) circulation plays a key role in cerebral waste clearance via the glymphatic system. Although CSF flow velocity is an essential component of CSF dynamics, it has not been sufficiently characterized, and particularly, in studies of the glymphatic system in rat. To investigate the relationship between the flow velocity of CSF in the brain aqueduct and the glymphatic waste clearance rate, using phase-contrast MRI we performed the first measurements of CSF velocity in rats. Phase-contrast MRI was performed using a 7 T system to map mean velocity of CSF flow in the aqueduct in rat brain. The effects of age (3 months old versus 18 months old), gender, strain (Wistar, RNU, Dark Agouti), anesthetic agents (isoflurane versus dexmedetomidine), and neurodegenerative disorder (Alzheimer’ disease in Fischer TgF344-AD rats, males and females) on CSF velocity were investigated in eight independent groups of rats (12 rats per group). Our results demonstrated that quantitative velocities of CSF flow in the aqueduct averaged 5.16 ± 0.86 mm/s in healthy young adult male Wistar rats. CSF flow velocity in the aqueduct was not altered by rat gender, strain, and the employed anesthetic agents in all rats, also age in the female rats. However, aged (18 months) Wistar male rats exhibited significantly reduced the CSF flow velocity in the aqueduct (4.31 ± 1.08 mm/s). In addition, Alzheimer's disease further reduced the CSF flow velocity in the aqueduct of male and female rats.

Background: Stroke-induced neurogenesis and oligodendrogenesis contribute to improvement of neurological function after stroke. However, mechanisms underlying post stroke neurogenesis and oligodendrogenesis warrant investigation. Argonaute (Ago) genes, the major components of the RNA-induced silencing complex, regulate microRNA (miRNA) function for post-transcriptional gene silencing. The present study investigated the role of neural stem cell (NSC) specific Ago2 after stroke. Methods and Results: Compared to non-stroke NSCs, stroke significantly upregulated Ago2 expression in subventricular zone (SVZ) NSCs. Using adult male mice with conditional ablation of Ago2 in Ascl1 lineage NSCs (Ago2 cKO), we then examined NSC function in neurogenic regions of the SVZ and hippocampus. Under non-ischemic conditions, compared to wild-type littermates (WT), Ago2 cKO mice showed significantly reduced neuronal and oligodendrocyte differentiation of NSCs measured by newly generated neuroblasts (BrdU + /DCX + , 12±7/mm 2 cKO vs 32±11/mm 2 WT, n=3/group, p<0.05) in the neurogenic areas and new mature oligodendrocytes (BrdU + /CC1 + , 20±4/mm 2 cKO vs 40±16/mm 2 WT) in the corpus callosum. In addition, Ago2 cKO mice exhibited the learning and memory impairments. Moreover, Ago2 cKO mice subjected to middle cerebral artery occlusion exhibited significantly reduced sensorimotor functions as measured by the adhesive test, foot-fault test, and modified neurological severity scores compared to WT ischemic mice. Mechanistically, mRNA and miRNA sequencing and bioinformatics analyses showed that ablation of NSC Ago2 deregulated many neurogenic genes involved in the sonic hedgehog (Shh), Notch and TGFβ signaling pathways, and altered Ago2-bound miRNAs that potentially target Shh (miR17-92 cluster), Notch (miR-124, miR-146a) and and TGFβ (miR-21, miR-200c) pathway genes, suggesting that Ago2 regulates neurogenesis and oligodendrogenesis via these miRNA-mRNA interactions. Conclusions: Our data demonstrate an essential role of Ago2 in adult neurogenesis and oligodendrogenesis, and also provide potential therapeutic targets of Ago2-bound miRNAs for improvement of neurological outcomes after stroke by enhancing NSC function.

BACKGROUND:The glymphatic system actively exchanges cerebrospinal fluid (CSF) and interstitial fluid (ISF) to eliminate toxic interstitial waste solutes from the brain parenchyma. Impairment of the glymphatic system has been linked to several neurological conditions. Glioblastoma, also known as Glioblastoma multiforme (GBM) is a highly aggressive form of malignant brain cancer within the glioma category. However, the impact of GBM on the functioning of the glymphatic system has not been investigated. Using dynamic contrast-enhanced magnetic resonance imaging (CE-MRI) and advanced kinetic modeling, we examined the changes in the glymphatic system in rats with GBM.METHODS:Dynamic 3D contrast-enhanced T1-weighted imaging (T1WI) with intra-cisterna magna (ICM) infusion of paramagnetic Gd-DTPA contrast agent was used for MRI glymphatic measurements in both GBM-induced and control rats. Glymphatic flow in the whole brain and the olfactory bulb was analyzed using model-derived parameters of arrival time, infusion rate, clearance rate, and residual that describe the dynamics of CSF tracer over time.RESULTS:3D dynamic T1WI data identified reduced glymphatic influx and clearance, indicating an impaired glymphatic system due to GBM. Kinetic modeling and quantitative analyses consistently indicated significantly reduced infusion rate, clearance rate, and increased residual of CSF tracer in GBM rats compared to control rats, suggesting restricted glymphatic flow in the brain with GBM. In addition, our results identified compromised perineural pathway along the optic nerves in GBM rats.CONCLUSIONS:Our study demonstrates the presence of GBM-impaired glymphatic response in the rat brain and impaired perineural pathway along the optic nerves. Reduced glymphatic waste clearance may lead to the accumulation of toxic waste solutes and pro-inflammatory signaling molecules which may affect the progression of the GBM.

BACKGROUND:Ischemic stroke affects about 700 000 patients per year in the United States, and to date, there are no effective pharmacological agents that promote recovery. Here, we studied the pharmacokinetics, pharmacodynamics, and efficacy of NTS-105, a novel neuroactive steroid, and NTS-104, a prodrug of NTS-105, in 2 models of ischemic stroke.METHODS:The pharmacodynamics and pharmacokinetics of NTS-104/105 were investigated in naive and stroke rats, and models of embolic and transient middle cerebral artery occlusion were used to investigate the dose-related effects of NTS-104. All rats were randomly assigned into the experimental groups, and all outcome measurements were performed blindly.RESULTS:Blood plasma and brain pharmacokinetic analysis revealed that NTS-104 rapidly converted to NTS-105, which reached peak concentration at ≈1 hour after dosing and distributed similarly to normal and ischemic brains. NTS-104 administration 4 hours after embolic middle cerebral artery occlusion led to a dose-dependent improvement of neurological outcomes and a dose-dependent reduction of infarct volumes relative to vehicle-treated animals. A single dose level study confirmed that NTS-104 administered 4 hours after transient middle cerebral artery occlusion was also neuroprotective. Quantitative ELISA revealed that NTS-104 treatment resulted in time- and dose-dependent changes in AKT activation and cytokine levels within the ischemic brain, which included reductions of IL-6, VEGF, ICAM-1, IL-1β, MCP-1, RAGE, and GM-CSF. Time- and dose-dependent reductions in IL-6 and GM-CSF were also observed in the plasma along with an elevation of galectin-1.CONCLUSIONS:NTS-104 is a novel prodrug that converts to a novel neuroactive steroid, NTS-105, which improves functional outcomes in experimental ischemic stroke models.

Introduction: Stroke-induced oligodendrogenesis contributes to ischemic brain remodeling and spontaneous functional recovery. Genes that regulate microRNA biogenesis in stroke-induced oligodendrogenesis remain little known. Methods: We employed a transgenic mouse line with conditional and inducible ablation of Dicer, a key gene of miRNA biogenesis, in myelin proteolipid protein (PLP) lineage oligodendrocytes (Dicer-KO). A PLP reporter mouse line was used as a control. Mice were subjected to transient (60min) middle cerebral artery occlusion (MCAO, n=8/group). An array of behavioral and cognitive tests were performed. All mice were sacrificed at 28 days after MCAO. Fluorescence-activated cell sorting (FACS) in combination with miRNA sequencing were used to profile the miRNA alteration. Results: Compared to ischemic reporter mice, ischemic Dicer-KO mice exhibited significantly increased infarct volumes and significantly impaired neurological outcomes and cognitive deficits post stroke as assayed by social recognition memory, novel object recognition, and Morris water-maze tests. Immunochemistry analysis revealed that ischemic Dicer-KO mice showed a significant reduction of the number of PLP-lineage NG2 + oligodendrocyte precursor cells (OPCs) and APC + myelinating oligodendrocytes compared to ischemic reporter mice. Moreover, Dicer-KO mice exhibited a robust reduction of myelinated axons measured by myelin binding protein (MBP) and Bielschowsky/Luxol Fast Blue assay. FACS analysis of PLP-lineage cells in peri-infarct regions showed that ablation of Dicer significantly decreased many miRNAs including the miR-200 family that are known to regulate oligodendrogenesis. Conclusion: Our data indicate that Dicer in PLP lineage oligodendrocytes is required for stroke-induced oligodendrogenesis and myelination, and that Dicer is involved in spontaneous functional recovery after stroke.

Ongoing neurovascular dysfunction contributes to type 2 diabetes mellitus (T2DM)-induced cognitive deficits. However, it is not known whether early post onset of T2DM interventions may reduce evolving neurovascular dysfunction and thereby lead to diminution of T2DM-induced cognitive deficits. Using multiple MRI metrics, we evaluated neurovascular changes in T2DM rats treated with exosomes derived from cerebral endothelial cells (CEC-Exos). Two months after induction of T2DM in middle-aged male rats by administration of streptozotocin nicotinamide, rats were randomly treated with CEC-Exos twice weekly or saline for 4 consecutive weeks (n = 10/group). MRI measurements were performed at the end of the treatment, which included cerebral blood flow (CBF), contrast-enhanced T-1-weighted imaging, and relaxation time constants T-1 and T-2. MRI analysis showed that compared with controls, the CEC-Exo-treated T2DM rats exhibited significant elevation of T-2 and CBF in white matter and significant augmentation of T-1 and reduction of blood-brain barrier permeability in gray matter. In the hippocampus, CEC-Exo treatment significantly increased T-1 and CBF. Furthermore, CEC-Exo treatment significantly reduced T2DM-induced cognitive deficits measured by the Morris water maze and odor recognition tests. Collectively, our corresponding MRI data demonstrate that treatment of T2DM rats with CEC-Exos robustly reduced neurovascular dysfunction in gray and white matter and the hippocampus.

Neurogenesis contributes to poststroke recovery. Long noncoding RNAs (lncRNAs) participate in the regulation of stem cell self-renewal and differentiation. However, the role of lncRNAs in stroke-induced neurogenesis remains unknown. In this study, we found that H19 was the most highly upregulated lncRNA in neural stem cells (NSCs) of the subventricular zone (SVZ) of rats subjected to focal cerebral ischemia. Deletion of H19 suppressed cell proliferation, promoted cell death, and blocked NSC differentiation. RNA sequencing analysis revealed that genes deregulated by H19 knockdown were those that are involved in transcription, apoptosis, proliferation, cell cycle, and response to hypoxia. H19 knockdown significantly increased the transcription of cell cycle-related genes including p27, whereas overexpression of H19 substantially reduced expression of these genes through the interaction with chromatin remodeling proteins EZH2 and SUZ12. Moreover, H19 regulated neurogenesis-related miRNAs. Inactivation of H19 in NSCs of ischemic rats attenuated spontaneous functional recovery after stroke. Collectively, our data provide novel insights into the epigenetic regulation of lncRNAs in stroke-induced neurogenesis.

Normal aging is a risk factor for metabolic disorders such as diabetes, and diabetes is also a recognized cause of accelerated aging. Being able to distinguish changes caused by normal aging from those caused by diabetes, would provide insight into how the aging brain interacts with diabetes. Eight types of MRI metric maps (magnetization relaxation time constants of T1 and T2, cerebral blood flow, cerebrovascular permeability, mean diffusivity, diffusion fractional anisotropy, mean diffusion kurtosis and diffusion directional entropy) were generated for all rats from the three groups of normal young, healthy and 1.5-month diabetic middle-aged rats under investigation. Measurements of multiple MRI indices of cerebral white and gray matter from animals of the three groups provide complementary results and insight into differences between healthy and diabetic white / gray matter in the mid-aged rats. Our data indicate that MRI may distinguish between the normal and diabetes in mid-aged rat brains by measuring either T1 and T2 of gray matter, or fractional anisotropy of white matter and gray matter. Therefore, MRI can distinguish changes of cerebral tissue due to the normal aging from diabetic aging, which may lead to be able to better understand how diabetes accelerates aging in normal brain.

Introduction: The hypothalamus regulates the neuroendocrine system. Hypothalamic neurogenesis is localized to the arcuate nucleus (AR) close to the third ventricle (3V) and to the bottom of the 3V in the median eminence (ME). Molecular mechanisms, in particular miRNAs, in regulating hypothalamic neurogenesis remain unknown. Dicer processes precursor miRNAs into mature miRNAs. Using a transgenic mouse line with an inducible and conditional ablation of Dicer in adult neural stem cells (NST), we tested the hypothesis that Dicer regulates stroke-increased hypothalamic neurogenesis. Methods and Results: Using a NST reporter mouse line (Ascl1-CreERT2:Tomatoflox/flox ), we found that NST were present in the AR and the ME. Tamoxifen was administered to mice (n=32) with conditional ablation of Dicer (Dicer/Cko) in NTSs (Ascl1-CreERT2:Dicerflox/flox) to knockout Dicer. Mice were then subjected to focal cerebral ischemia and wild-type mice (n=26) were used as control. Double and triple immunofluorescent staining with 3D confocal microscopic analysis was performed to quantify proliferation and phenotypes of NST. In wild-type mice, stroke significantly (P<0.05) increased BrdU+ NST in the AR (52 ± 3 cells/mm2 vs 40 ± 4 no-stroke) and the ME (45 cells/mm2 ± 4 vs 29 ± 4 no-stroke) 14 days after stroke. The increased proliferating NST were associated with substantial augmentation of newly generated neurons (BrdU+/NeuN+ cells) in the AR (6 ± 1% vs 3 ± 0.4%, no-stroke) and in the ME (5 ± 1% vs 2 ± 0.3%, no-stroke) 30 days after stroke. These data indicate that stroke increases hypothalamic neurogenesis. However, compared to wild-type ischemic mice, ischemic Dicer/Cko mice exhibited significant (p<0.01) reduction of BrdU+ NTS in the AR (41 ± 4) and in the ME (33 ± 4) 14 days after stroke, and significant (p<0.01) decreases of newly generated neurons in the AR (4 ± 0.6%) and in the ME (3 ± 0.5%) 30 days after stroke. Ablation of Dicer in NTS of mice without stroke also resulted in significant reduction of proliferating NST (23 ± 2 cells/mm2 AR, 17 ± 3 ME) and newly generated neurons (2 ± 0.4% AR, 1 ± 0.2 ME) compared to non-ischemic wild-type mice. Conclusion: Our data provide first evidence that Dicer regulates adult hypothalamic neurogenesis under physiological and ischemic conditions.

Introduction: Diabetes mellitus (DM) is associated with cognitive decline and dementia in the elderly. The glymphatic system mediates clearance of the interstitial solutes in the brain by exchange of cerebrospinal and interstitial fluid (CSF and ISF). We recently demonstrated that DM in aged rat induces impairment of the glymphatic system and cognitive decline. Exosomes, membrane vesicles, mediate intercellular communication by transferring their cargo into recipient cells. The present study investigated whether cerebral endothelial exosomes (CEE) ameliorate glymphatic system impairment and improve cognitive function in aged DM rats. Methods and Results: DM was induced in male Wistar rats (13 months, n=48) by injection of nicotinamide and streptozotocin. Two months after DM, rats were treated with CEE (1x10 11 exosomes/rat, IV) twice a week for 4 weeks. Age matched DM and non-DM rats were used as controls. CEE were harvested from the cultured cerebral endothelial cells of health young adult rats. Exchanges of CSF and ISF were measured by intracisternal injection of fluorescent tracer, Texas Red-dextran (TR, 3kD). Confocal microscopic analysis of brain slices revealed a progressive slowdown of ISF clearance in the hippocampi, assessed by retention of TR starting at 2.5 fold at 2M (13±5 vs 5±3% of area) and increasing to 4 fold at 4M (21±4 vs 5±2%) of DM. Paravascular amyloid β (Aβ) accumulation was only detected at 4M of DM. The CEE treatment significantly (p<0.05) reduced TR retention (10±4%) at 4M of DM and also decreased Aβ accumulation (2±1 vs 6±2/mm 2 ) and parenchymal fibrin deposition (9±5 vs 23±5/mm 2 ) compared to untreated DM rats. Moreover, the CEE treatment significantly improved hippocampal related learning and memory measured by the Morris Water Maze and odor-based novelty recognition for olfactory memory, without altering the glucose level. In vitro, cerebral endothelial cells isolated from 2M DM rats exhibited substantial dysfunction as measured by capillary-like tube formation and cell migration, whereas incubation with the CEE substantially reversed endothelial dysfunction. Conclusions: The CEE treatment reduces DM-induced glymphatic and cerebral endothelial dysfunctions, leading to improvement of cognitive function in aged DM rats.

Background: MicroRNAs regulate adult neurogenesis. Conditional deletion of Dicer in neural stem cells (NSCs) causes postnatal death. The functional role of Dicer in adult neurogenesis remains unknown. Using mice with inducible conditional knock-down of Dicer in adult NSCs, we investigated the effect of ablation of Dicer on neurogenesis and cognitive function. Methods and Results: Young adult Ascl1-CreER:Dicerflox/flox mice (Dicer Cko, n= 32) were administered (i.p) with tamoxifen daily for 5 consecutive days and age matched wild-type litters (n= 30) were used as control. The mice were sacrificed at 2, 14 or 30 days after injection. Immunstaining was performed to detect phenotypes of subventricular zone (SVZ) cells. An array of cognitive tests including Morris Water Maze, odor-based novelty recognition, and sociability test were performed. Primary NPCs were isolated from the SVZ for vitro studies. Compared to control, Dicer CKo had 54% reduction of Dicer protein in NPCs. Cognitive tests showed that CKo spent 35% less time in the correct quadrant (p<0.05) of Morris Water Maze, significantly reduced time exploring new odor objects, 44 ±10% (p<0.05) in Cko animals compared with 77 ±10% in control, and significantly less time with other mice when they encountered a strange mouse during the sociability test (70 ± 9 vs 118 ±7 seconds, p<0.05). CKo significantly (p<0.05) reduced BrdU+ (16±2% vs 24±3%), and Ki67+ NPCs (25±3% vs 33±3%), doublecortin (DCX)+ neuroblasts (2± 0.6% vs 6± 1%), and Ng2+ oligodendrocyte progenitor cells (OPCs, 8±1% vs 12± 2%) in the SVZ. However, Dicer CKo animals exhibited a significant increase (p<0.05) of apoptotic NPCs (14±0.5% vs 10 ±0.3%). These in vivo findings were consistent with data from cultured NPCs. Dicer CKo also showed significant (p<0.05) reduction DCX+ neuroblasts in the rostral migratory stream and APC+ mature oligodendrocytes in the corpus callosum. Conclusion: Our data demonstrated that inducible conditional ablation of Dicer in Ascl1 lineage NPCs impairs neurogenesis and oligodendrogenesis in adult SVZ niche and white matter, and induces cognitive impairment, indicating that Dicer in adult NPCs is essential for maintaining neurogenesis and oligodendrogenesis and is important for cognitive function.

Background: Adult neural stem cells (NSCs) are present in the rodent ventricular-subventricular zone (V/SVZ) of the lateral ventricles. However, their in vivo role in stroke-induced neurogenesis remains uncertain. Using an anti-mitotic agent (Ara-C) in combination with phenotype markers, we examined NSCs in whole-mount preparation of the lateral ventricle of ischemic brain. Methods and Results: Adult mice (n=28) were used. Focal cerebral ischemia was induced by occluding the right middle cerebral artery. Ara-C was infused on brain surface of mice for 7 days. BrdU was administered to label proliferating cells. Another 6 mice with non-ischemia, non-infusion were also given BrdU labeling as normal control. The architecture of NSCs identified by inhibitor of DNA binding/Differentiation 1 (Id1, a transcription factor)+/GFAP+ in the V/SVZ were examined in whole mount by means of a 3D confocal microscope. In a normal V/SVZ, 3% (at least 4,000 cells counted per each sample) cells were Id1+. Of these Id1+ cells, 16% cells were GFAP+, which were anchored by specialized apical end-feet in the center of the pinwheel of ependymal cells and they often extended basal processes at least 50 -80 um long; none of these cells were BrdU+ after 1h pulse labeling, indicating that these cells are relatively quiescent. However, 69 % of Id1+ cells were Mash+ and Olig2+ and the majority of these cells were BrdU+, indicating that these are active progenitor cells. 1h after termination of infusion, Id1+/Mash+ and Olig2+ cells were eliminated, whereas Id1+/GFAP+ cells in the center of pinwheels were not affected in non-ischemic and ischemic V/SVZ niches. At this time point, there were no actively proliferating cells measured by 1h BrdU pulse labeling. However, by 48h after termination of infusion, there were many proliferating cells with 59% and 75% of BrdU+ cells were Id1+ in non-ischemic and ischemic V/SVZ niches (p<0.05), respectively. In the ischemic V/SVZ niche, in the center of pinwheels the majority of Id1+/BrdU+ cells were GFAP+ . Conclusion: Our in vivo data suggest that adult V/SVZ niche GFAP+/Id1+ astrocytes are “functional quiescent” NSCs. Stroke recruits quiescent NSCs in the V/SVZ niche to an active pool for enhancing the neurogenic process in response to ischemic insult.

Background: The MicroRNA 17-92 (miR17-92) cluster regulates endothelial homeostasis. However, the effect of the miR17-92 cluster in cerebral endothelial cells on blood brain barrier (BBB) has not been investigated. We examined BBB integrity in a transgenic mouse line in which miR17-92 cluster is ablated in endothelial cells. Methods and Results: Adult Tie2-Cre:miR17-92 flox/flox (Tie2/miR17-92) mice and Tie2-Cre:Tomato (Tie2/red) reporter mice were used. Cerebral vessels in Tie2/red reporter mice exhibited red fluorescence, indicating specific expression of Tie2 in cerebral endothelial cells. RT-PCR analysis revealed ~70% reduction of miRNA levels of individual members of the miR17-92 cluster in endothelial cells harvested from Tie2/miR17-92 mouse brain, confirming deletion of the miR17-92 cluster. Time-lapse microscopic analysis of cultured endothelial cells showed a substantial reduction of capillary formation by miR17-92 cluster knockout endothelial cells compared to the cells harvested from Tie2/red reporter mice. In vivo, analysis of 3D cerebral microvessels showed that ablation of the miR17-92 markedly (P<0.05) increased capillary diameter in the cortex (7.1±0.4μm vs 5.1±0.3μm in reporter, n=4 mice/group), corpus callosum (CC, 6.6±0.4 vs 4.7±0.3), and striatum (6.7±0.4 vs 5.0±0.3). Triple immunofluorescent staining showed that the absence of this cluster substantially (p<0.05) reduced pericytes identified by platelet-derived growth factor-β positive cells (32±6 cells vs 80±6 cells in cortex, 34±7 vs 77±6 in CC, and 36±6 vs 79±5 in striatum) and decreased astrocyte end-foot processes identified by aquaporin 4 positive cells (42±6 cells vs 72±6 cells in cortex, 40±10 vs 70±6 in CC, and 36±6 vs 65±8 in striatum). These data suggest that cerebral endothelil cells lacking miR17-92 causes disruption of vascular coverage by pericytes and astrocyte end-foot processes. Deletion of the miR17-92 cluster also increased extravasation of Evans blue dye into the parenchyma and reduced the number of doublecortin positive neuroblasts in the subventricular zone. Conclusion: Our data indicate that ablation of the miR17-92 cluster in cerebral endothelial cells disrupts BBB integrity, which could exacerbate ischemic damage.

The unique cellular and vascular architecture of the adult ventricular-subventricular zone (V/SVZ) neurogenic niche plays an important role in regulating neural stem cell function. However, the in vivo identification of neural stem cells and their relationship to blood vessels within this niche in response to stroke remain largely unknown. Using whole-mount preparation of the lateral ventricle wall, we examined the architecture of neural stem cells and blood vessels in the V/SVZ of adult mouse over the course of 3 months after onset of focal cerebral ischemia. Stroke substantially increased the number of glial fibrillary acidic protein (GFAP) positive neural stem cells that are in contact with the cerebrospinal fluid (CSF) via their apical processes at the center of pinwheel structures formed by ependymal cells residing in the lateral ventricle. Long basal processes of these cells extended to blood vessels beneath the ependymal layer. Moreover, stroke increased V/SVZ endothelial cell proliferation from 2% in non-ischemic mice to 12 and 15% at 7 and 14 days after stroke, respectively. Vascular volume in the V/SVZ was augmented from 3% of the total volume prior to stroke to 6% at 90 days after stroke. Stroke-increased angiogenesis was closely associated with neuroblasts that expanded to nearly encompass the entire lateral ventricular wall in the V/SVZ. These data indicate that stroke induces long-term alterations of the neural stem cell and vascular architecture of the adult V/SVZ neurogenic niche. These post-stroke structural changes may provide insight into neural stem cell mediation of stroke-induced neurogenesis through the interaction of neural stem cells with proteins in the CSF and their sub-ependymal neurovascular interaction.

Stroke induces angiogenesis in the peri-infarct region. It is not known whether angiogenesis occurs in the subventricular zone (SVZ) of the lateral ventricle after stroke. The SVZ is a neural stem cell niche containing vascular plexus that supports adult neurogenesis. We characterized longitudinal changes of vascular structures in the SVZ niche after stroke using whole mounts, an organotypic preparation of the SVZ in which the 3D cell-vessel relationships are preserved. Adult mice were subjected to middle cerebral artery occlusion (MCAO). The vascular architectures within the 50 μm thick SVZ of immunostained whole mounts were imaged by 3D confocal microscopy. In non-MCAO mice (n=4), 2±0.2% of endothelial cells were proliferative (BrdU+/CD31+). Blood vessels in this niche constituted 2.6±0.01% of the total volume with 75±17 vascular branches. However, 7 and 14 days after MCAO, proliferated endothelial cells significantly (p<0.05) increased to 12±1% (n=5) and 15±1 % (n=5), respectively, which was followed by substantial increases in vascular volume at 14 (4.2±0.01%, n=3), 30 (4.9±0.05%, n=3), and 90 (5.7±0.01%, n=3) days, but not at 7 days after MCAO. Moreover, vascular branches increased significantly to 156±27 and 216±8 at 30 and 90 days, respectively, but not at 14 days. Interestingly, we detected increases in the number of string-like vessels starting at 14 days (731±79/mm 3 vs 476±41/mm 3 in control) which increased and persisted at 30 (1,824±255/mm 3 ) and 90 (1,748±204/mm 3 ) days after MCAO. These string-like vessels were not perfused by plasma. String vessels increase during embryonic angiogenesis. Collectively, these data indicate that stroke induces angiogenesis in the SVZ, which lasts at least 90 days after stroke. Moreover, stroke significantly increased neural stem cells (BrdU + /GFAP + , 13±3%, 15±3%, and 11±4% at 7, 14, and 90 days, respectively, vs 6±1% in control) and newborn neurons (BrdU + /DCX + , 14±2% and 12±2.0% at 7 and 14 days, respectively, vs 4±1% in control). Neural stem cells at the ventricular surface extended their processes to the blood vessels in the SVZ. Our data indicate that stroke induces angiogenesis in the SVZ, which is associated with stroke-induced neurogenesis.

Adult neural stem cells give rise to neurons, oligodendrocytes and astrocytes. Aging reduces neural stem cells. Using an inducible nestin-CreER(T2)/R26R-yellow fluorescent protein (YFP) mouse, we investigated the effect of Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, on nestin lineage neural stem cells and their progeny in the ischemic brain of the middle-aged mouse. We showed that focal cerebral ischemia induced nestin lineage neural stem cells in the subventricular zone (SVZ) of the lateral ventricles and nestin expressing NeuN positive neurons and adenomatous polyposis coli (APC) positive mature oligodendrocytes in the ischemic striatum and corpus callosum in the aged mouse. Treatment of the ischemic middle-aged mouse with Sildenafil increased nestin expressing neural stem cells, mature neurons, and oligodendrocytes by 33, 75, and 30%, respectively, in the ischemic brain. These data indicate that Sildenafil amplifies nestin expressing neural stem cells and their neuronal and oligodendrocyte progeny in the ischemic brain of the middle-aged mouse.