The Association between Glymphatic System and Perivascular Macrophages in Brain Waste Clearance.

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.

The Association between Glymphatic System and Perivascular Macrophages in Brain Waste Clearance.

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 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.

Early therapeutic effects of an Angiopoietin-1 mimetic peptide in middle-aged rats with vascular dementia.

Background: Vascular Dementia (VaD) refers to dementia caused by cerebrovascular disease and/or reduced blood flow to the brain and is the second most common form of dementia after Alzheimer's disease. We previously found that in middle-aged rats subjected to a multiple microinfarction (MMI) model of VaD, treatment with AV-001, a Tie2 receptor agonist, significantly improves short-term memory, long-term memory, as well as improves preference for social novelty compared to control MMI rats. In this study, we tested the early therapeutic effects of AV-001 on inflammation and glymphatic function in rats subjected to VaD. Methods: Male, middle-aged Wistar rats (10-12 m), subjected to MMI, were randomly assigned to MMI and MMI + AV-001 treatment groups. A sham group was included as reference group. MMI was induced by injecting 800 ± 200, 70-100 µm sized, cholesterol crystals into the internal carotid artery. Animals were treated with AV-001 (1 µg/Kg, i.p.) once daily starting at 24 h after MMI. At 14 days after MMI, inflammatory factor expression was evaluated in cerebrospinal fluid (CSF) and brain. Immunostaining was used to evaluate white matter integrity, perivascular space (PVS) and perivascular Aquaporin-4 (AQP4) expression in the brain. An additional set of rats were prepared to test glymphatic function. At 14 days after MMI, 50 µL of 1% Tetramethylrhodamine (3 kD) and FITC conjugated dextran (500 kD) at 1:1 ratio were injected into the CSF. Rats (4-6/group/time point) were sacrificed at 30 min, 3 h, and 6 h from the start of tracer infusion, and brain coronal sections were imaged using a Laser scanning confocal microscope to evaluate tracer intensities in the brain. Result: Treatment of MMI with AV-001 significantly improves white matter integrity in the corpus callosum at 14 days after MMI. MMI induces significant dilation of the PVS, reduces AQP4 expression and impairs glymphatic function compared to Sham rats. AV-001 treatment significantly reduces PVS, increases perivascular AQP4 expression and improves glymphatic function compared to MMI rats. MMI significantly increases, while AV-001 significantly decreases the expression of inflammatory factors (tumor necrosis factor-α (TNF-α), chemokine ligand 9) and anti-angiogenic factors (endostatin, plasminogen activator inhibitor-1, P-selectin) in CSF. MMI significantly increases, while AV-001 significantly reduces brain tissue expression of endostatin, thrombin, TNF-α, PAI-1, CXCL9, and interleukin-6 (IL-6). Conclusion: AV-001 treatment of MMI significantly reduces PVS dilation and increases perivascular AQP4 expression which may contribute to improved glymphatic function compared to MMI rats. AV-001 treatment significantly reduces inflammatory factor expression in the CSF and brain which may contribute to AV-001 treatment induced improvement in white matter integrity and cognitive function.

Therapeutic effects of CD133 + Exosomes on liver function after stroke in type 2 diabetic mice.

Background and purpose: Non-alcoholic fatty liver disease (NAFLD) is known to adversely affect stroke recovery. However, few studies investigate how stroke elicits liver dysfunction, particularly, how stroke in type 2 diabetes mellitus (T2DM) exacerbates progression of NAFLD. In this study, we test whether exosomes harvested from human umbilical cord blood (HUCBC) derived CD133 + cells (CD133 + Exo) improves neuro-cognitive outcome as well as reduces liver dysfunction in T2DM female mice. Methods: Female, adult non-DM and T2DM mice subjected to stroke presence or absence were considered. T2DM-stroke mice were randomly assigned to receive PBS or Exosome treatment group. CD133 + Exo (20 µg/200 µl PBS, i.v.) was administered once at 3 days after stroke. Evaluation of neurological (mNSS, adhesive removal test) and cognitive function [novel object recognition (NOR) test, odor test] was performed. Mice were sacrificed at 28 days after stroke and brain, liver, and serum were harvested. Results: Stroke induces severe and significant short-term and long-term neurological and cognitive deficits which were worse in T2DM mice compared to non-DM mice. CD133 + Exo treatment of T2DM-stroke mice significantly improved neurological function and cognitive outcome indicated by improved discrimination index in the NOR and odor tests compared to control T2DM-stroke mice. CD133 + Exo treatment of T2DM stroke significantly increased vascular and white matter/axon remodeling in the ischemic brain compared to T2DM-stroke mice. However, there were no differences in the lesion volume between non-DM stroke, T2DM-stroke and CD133 + Exo treated T2DM-stroke mice. In T2DM mice, stroke induced earlier and higher TLR4, NLRP3, and cytokine expression (SAA, IL1ß, IL6, TNFα) in the liver compared to heart and kidney, as measured by Western blot. T2DM-stroke mice exhibited worse NAFLD progression with increased liver steatosis, hepatocellular ballooning, fibrosis, serum ALT activity, and higher NAFLD Activity Score compared to T2DM mice and non-DM-stroke mice, while CD133 + Exo treatment significantly attenuated the progression of NAFLD in T2DM stroke mice. Conclusion: Treatment of female T2DM-stroke mice with CD133 + Exo significantly reduces the progression of NAFLD/NASH and improves neurological and cognitive function compared to control T2DM-stroke mice.

Post-Stroke Administration of L-4F Promotes Neurovascular and White Matter Remodeling in Type-2 Diabetic Stroke Mice.

Patients with type 2 diabetes mellitus (T2DM) exhibit a distinct and high risk of ischemic stroke with worse post-stroke neurovascular and white matter (WM) prognosis than the non-diabetic population. In the central nervous system, the ATP-binding cassette transporter member A 1 (ABCA1), a reverse cholesterol transporter that efflux cellular cholesterol, plays an important role in high-density lipoprotein (HDL) biogenesis and in maintaining neurovascular stability and WM integrity. Our previous study shows that L-4F, an economical apolipoprotein A member I (ApoA-I) mimetic peptide, has neuroprotective effects via alleviating neurovascular and WM impairments in the brain of db/db-T2DM stroke mice. To further investigate whether L-4F has neurorestorative benefits in the ischemic brain after stroke in T2DM and elucidate the underlying molecular mechanisms, we subjected middle-aged, brain-ABCA1 deficient (ABCA1-B/-B), and ABCA1-floxed (ABCA1fl/fl) T2DM control mice to distal middle cerebral artery occlusion. L-4F (16 mg/kg, subcutaneous) treatment was initiated 24 h after stroke and administered once daily for 21 days. Treatment of T2DM-stroke with L-4F improved neurological functional outcome, and decreased hemorrhage, mortality, and BBB leakage identified by decreased albumin infiltration and increased tight-junction and astrocyte end-feet densities, increased cerebral arteriole diameter and smooth muscle cell number, and increased WM density and oligodendrogenesis in the ischemic brain in both ABCA1-B/-B and ABCA1fl/fl T2DM-stroke mice compared with vehicle-control mice, respectively (p < 0.05, n = 9 or 21/group). The L-4F treatment reduced macrophage infiltration and neuroinflammation identified by decreases in ED-1, monocyte chemoattractant protein-1 (MCP-1), and toll-like receptor 4 (TLR4) expression, and increases in anti-inflammatory factor Insulin-like growth factor 1 (IGF-1) and its receptor IGF-1 receptor ß (IGF-1Rß) in the ischemic brain (p < 0.05, n = 6/group). These results suggest that post-stroke administration of L-4F may provide a restorative strategy for T2DM-stroke by promoting neurovascular and WM remodeling. Reducing neuroinflammation in the injured brain may contribute at least partially to the restorative effects of L-4F independent of the ABCA1 signaling pathway.

Treatment With an Angiopoietin-1 Mimetic Peptide Improves Cognitive Outcome in Rats With Vascular Dementia.

Background and Purpose: Vascular dementia (VaD) is a complex neurodegenerative disease affecting cognition and memory. There is a lack of approved pharmacological treatments specifically for VaD. In this study, we investigate the therapeutic effects of AV-001, a Tie2 receptor agonist, in middle-aged rats subjected to a multiple microinfarct (MMI) model of VaD. Methods: Male, 10-12 month-old, Wistar rats were employed. The following experimental groups were used: Sham, MMI, MMI+1 µg/Kg AV-001, MMI+3 µg/Kg AV-001, MMI+6 µg/Kg AV-001. AV-001 treatment was initiated at 1 day after MMI and administered once daily via intraperitoneal injection. An investigator blinded to the experimental groups conducted a battery of neuro-cognitive tests including modified neurological severity score (mNSS) test, novel object recognition test, novel odor recognition test, three chamber social interaction test, and Morris water maze test. Rats were sacrificed at 6 weeks after MMI. Results: There was no mortality observed after 1, 3, or 6 µg/Kg AV-001 treatment in middle-aged rats subjected to MMI. AV-001 treatment (1, 3, or 6 µg/Kg) does not significantly alter blood pressure or heart rate at 6 weeks after MMI compared to baseline values or the MMI control group. Treatment of MMI with 1 or 3 µg/Kg AV-001 treatment does not significantly alter body weight compared to Sham or MMI control group. While 6 µg/Kg AV-001 treated group exhibit significantly lower body weight compared to Sham and MMI control group, the weight loss is evident starting at 1 day after MMI when treatment was initiated and is not significantly different compared to its baseline values at day 0 or day 1 after MMI. AV-001 treatment significantly decreases serum alanine aminotransferase, serum creatinine, and serum troponin I levels compared to the MMI control group; however, all values are within normal range. MMI induces mild neurological deficits in middle-aged rats indicated by low mNSS scores (<6 on a scale of 0-18). Compared to control MMI group, 1 µg/Kg AV-001 treatment group did not exhibit significantly different mNSS scores, while 3 and 6 µg/Kg AV-001 treatment induced significantly worse mNSS scores on days 21-42 and 14-42 after MMI, respectively. MMI in middle-aged rats induces significant cognitive impairment including short-term memory loss, long-term memory loss, reduced preference for social novelty and impaired spatial learning and memory compared to sham control rats. Rats treated with 1 µg/Kg AV-001 exhibit significantly improved short-term and long-term memory, increased preference for social novelty, and improved spatial learning and memory compared to MMI rats. Treatment with 3 µg/Kg AV-001 improves short-term memory and preference for social novelty but does not improve long-term memory or spatial learning and memory compared to MMI rats. Treatment with 6 µg/Kg AV-001 improves only long-term memory compared to MMI rats. Thus, 1 µg/Kg AV-001 treatment was selected as an optimal dose. Treatment of middle-aged rats subjected to MMI with 1 µg/Kg AV-001 significantly increases axon density, myelin density and myelin thickness in the corpus callosum, as well as increases synaptic protein expression, neuronal branching and dendritic spine density in the cortex, oligodendrocytes and oligodendrocyte progenitor cell number in the cortex and striatum and promotes neurogenesis in the subventricular zone compared to control MMI rats. Conclusions: In this study, we present AV-001 as a novel therapeutic agent to improve cognitive function and reduce white matter injury in middle aged-rats subjected to a MMI model of VaD. Treatment of MMI with 1 µg/Kg AV-001 significantly improves cognitive function, and increases axon density, remyelination and neuroplasticity in the brain of middle-aged rats.

Deficiency of Endothelial Nitric Oxide Synthase (eNOS) Exacerbates Brain Damage and Cognitive Deficit in A Mouse Model of Vascular Dementia.

Vascular Dementia (VaD) accounts for nearly 20% of all cases of dementia. eNOS plays an important role in neurovascular remodeling, anti-inflammation, and cognitive functional recovery after stroke. In this study, we investigated whether eNOS regulates brain damage, cognitive function in mouse model of bilateral common carotid artery stenosis (BCAS) induced VaD. Late-adult (6-8 months) C57BL/6J and eNOS knockout (eNOS-/-) mice were subjected to BCAS (n=12/group) or sham group (n=8/group). BCAS was performed by applying microcoils to both common carotid arteries. Cerebral blood flow (CBF) and blood pressure were measured. A battery of cognitive functional tests was performed, and mice were sacrificed 30 days after BCAS. Compared to corresponding sham mice, BCAS in wild-type (WT) and eNOS-/- mice significantly: 1) induces short term, long term memory loss, spatial learning and memory deficits; 2) decreases CBF, increases ischemic cell damage, including apoptosis, white matter (WM) and axonal damage; 3) increases blood brain barrier (BBB) leakage, decreases aquaporin-4 (AQP4) expression and vessel density; 4) increases microglial, astrocyte activation and oxidative stress in the brain; 5) increases inflammatory factor interleukin-1 receptor-associated kinase-1(IRAK-1) and amyloid beta (Aß) expression in brain; 6) increases IL-6 and IRAK4 expression in brain. eNOS-/-sham mice exhibit increased blood pressure, decreased iNOS and nNOS in brain compared to WT-sham mice. Compared to WT-BCAS mice, eNOS-/-BCAS mice exhibit worse vascular and WM/axonal damage, increased BBB leakage and inflammatory response, increased cognitive deficit, decreased iNOS, nNOS in brain. eNOS deficit exacerbates BCAS induced brain damage and cognitive deficit.

Cardiac Dysfunction in a Mouse Vascular Dementia Model of Bilateral Common Carotid Artery Stenosis.

Background: Cardiac function is associated with cognitive function. Previously, we found that stroke and traumatic brain injury evoke cardiac dysfunction in mice. In this study, we investigate whether bilateral common carotid artery stenosis (BCAS), a model that induces vascular dementia (VaD) in mice, induces cardiac dysfunction. Methods: Late-adult (6-8 months) C57BL/6J mice were subjected to sham surgery (n = 6) or BCAS (n = 8). BCAS was performed by applying microcoils (0.16 mm internal diameter) around both common carotid arteries. Cerebral blood flow and cognitive function tests were performed 21-28 days post-BCAS. Echocardiography was conducted in conscious mice 29 days after BCAS. Mice were sacrificed 30 days after BCAS. Heart tissues were isolated for immunohistochemical evaluation and real-time PCR assay. Results: Compared to sham mice, BCAS in mice significantly induced cerebral hypoperfusion and cognitive dysfunction, increased cardiac hypertrophy, as indicated by the increased heart weight and the ratio of heart weight/body weight, and induced cardiac dysfunction and left ventricular (LV) enlargement, indicated by a decreased LV ejection fraction (LVEF) and LV fractional shortening (LVFS), increased LV dimension (LVD), and increased LV mass. Cognitive deficits significantly correlated with cardiac deficits. BCAS mice also exhibited significantly increased cardiac fibrosis, increased oxidative stress, as indicated by 4-hydroxynonenal and NADPH oxidase-2, increased leukocyte and macrophage infiltration into the heart, and increased cardiac interleukin-6 and thrombin gene expression. Conclusions: BCAS in mice without primary cardiac disease provokes cardiac dysfunction, which, in part, may be mediated by increased inflammation and oxidative stress.

CD133+Exosome Treatment Improves Cardiac Function after Stroke in Type 2 Diabetic Mice.

Cardiac complications post-stroke are common, and diabetes exacerbates post-stroke cardiac injury. In this study, we tested whether treatment with exosomes harvested from human umbilical cord blood derived CD133+ cells (CD133+Exo) improves cardiac function in type 2 diabetes mellitus (T2DM) stroke mice. Adult (3-4 m), male, BKS.Cg-m+/+Leprdb/J (db/db, T2DM) and non-DM (db+) mice were randomized to sham or photothrombotic stroke groups. T2DM-stroke mice were treated with phosphate-buffered saline (PBS) or CD133+Exo (20 µg, i.v.) at 3 days after stroke. T2DM sham and T2DM+CD133+Exo treatment groups were included as controls. Echocardiography was performed, and mice were sacrificed at 28 days after stroke. Cardiomyocyte hypertrophy, myocardial capillary density, interstitial fibrosis, and inflammatory factor expression were measured in the heart. MicroRNA-126 expression and its target gene expression were measured in the heart. T2DM mice exhibit significant cardiac deficits such as decreased left ventricular ejection fraction (LVEF) and shortening fraction (LVSF), increased left ventricular diastolic dimension (LVDD), and reduced heart rate compared to non-DM mice. Stroke in non-DM and T2DM mice significantly decreases LVEF compared to non-DM and T2DM-sham, respectively. Cardiac dysfunction is worse in T2DM-stroke mice compared to non-DM-stroke mice. CD133+Exo treatment of T2DM-stroke mice significantly improves cardiac function identified by increased LVEF and decreased LVDD compared to PBS treated T2DM-stroke mice. In addition, CD133+Exo treatment significantly decreases body weight and blood glucose but does not decrease lesion volume in T2DM-stroke mice. CD133+Exo treatment of T2DM mice significantly decreases body weight and blood glucose but does not improve cardiac function. CD133+Exo treatment in T2DM-stroke mice significantly decreases myocardial cross-sectional area, interstitial fibrosis, transforming growth factor beta (TGF-ß), numbers of M1 macrophages, and oxidative stress markers 4-HNE (4-hydroxynonenal) and NADPH oxidase 2 (NOX2) in heart tissue. CD133+Exo treatment increases myocardial capillary density in T2DM-stroke mice as well as upregulates endothelial cell capillary tube formation in vitro. MiR-126 is highly expressed in CD133+Exo compared to exosomes derived from endothelial cells. Compared to PBS treatment, CD133+Exo treatment significantly increases miR-126 expression in the heart and decreases its target gene expression such as Sprouty-related, EVH1 domain-containing protein 1 (Spred-1), vascular cell adhesion protein (VCAM), and monocyte chemoattractant protein 1 (MCP1) in the heart of T2DM-stroke mice. CD133+Exo treatment significantly improves cardiac function in T2DM-stroke mice. The cardio-protective effects of CD133+Exo in T2DM-stroke mice may be attributed at least in part to increasing miR-126 expression and decreasing its target protein expression in the heart, increased myocardial capillary density and decreased cardiac inflammatory factor expression.

SUMO1 Deficiency Exacerbates Neurological and Cardiac Dysfunction after Intracerebral Hemorrhage in Aged Mice.

Small ubiquitin-like modifier 1 (SUMO1) reduces cardiac hypertrophy and induces neuroprotective effects. Previous studies have found that intracerebral hemorrhage (ICH) provokes cardiac deficit in the absence of primary cardiac diseases in mice. In this study, we tested the hypothesis that SUMO1 deficiency leads to worse brain and heart dysfunction after ICH and SUMO1 plays a key role in regulating brain-heart interaction after ICH in aged mice. Aged (18-20 months) female SUMO1 null (SUMO1-/-) mice and wild-type (WT) C57BL/6 J mice were randomly divided into four groups (n = 8/group): (1) WT-sham group, (2) SUMO1-/--sham group, (3) WT-ICH group, and (4) SUMO1-/--ICH group. Cardiac function was measured by echocardiography. Neurological and cognitive functional tests were performed. Mice were sacrificed at 10 days after ICH for histological and immunohistochemically staining. Compared with WT-sham mice, WT-ICH mice exhibited (1) significantly (P < 0.05) decreased SUMO1 expression in heart tissue, (2) evident neurological and cognitive dysfunction as well as brain white matter deficits, (3) significantly increased cardiac dysfunction, and (4) inflammatory factor expression in the heart and brain. Compared with WT-ICH mice, SUMO1-/--ICH mice exhibited significantly increased: (1) brain hemorrhage volume, worse neurological and cognitive deficits, and increased white matter deficits; (2) cardiac dysfunction and cardiac fibrosis; (3) inflammatory response both in heart and brain tissue. Aged SUMO1-deficient female mice subjected to ICH not only exhibit increased neurological and cognitive functional deficit but also significantly increased cardiac dysfunction and inflammatory cell infiltration into the heart and brain. These data suggest that SUMO1 plays an important role in brain-heart interaction.

Treatment with an Angiopoietin-1 mimetic peptide promotes neurological recovery after stroke in diabetic rats.

AIM: Vasculotide (VT), an angiopoietin-1 mimetic peptide, exerts neuroprotective effects in type one diabetic (T1DM) rats subjected to ischemic stroke. In this study, we investigated whether delayed VT treatment improves long-term neurological outcome after stroke in T1DM rats. METHODS: Male Wistar rats were induced with T1DM, subjected to middle cerebral artery occlusion (MCAo) model of stroke, and treated with PBS (control), 2 µg/kg VT, 3 µg/kg VT, or 5.5 µg/kg VT. VT treatment was initiated at 24 h after stroke and administered daily (i.p) for 14 days. We evaluated neurological function, lesion volume, vascular and white matter remodeling, and inflammation in the ischemic brain. In vitro, we evaluated the effects of VT on endothelial cell capillary tube formation and inflammatory responses of primary cortical neurons (PCN) and macrophages. RESULTS: Treatment of T1DM-stroke with 3 µg/kg VT but not 2 µg/kg or 5.5 µg/kg significantly improves neurological function and decreases infarct volume and cell death compared to control T1DM-stroke rats. Thus, 3 µg/kg VT dose was employed in all subsequent in vivo analysis. VT treatment significantly increases axon and myelin density, decreases demyelination, decreases white matter injury, increases number of oligodendrocytes, and increases vascular density in the ischemic border zone of T1DM stroke rats. VT treatment significantly decreases MMP9 expression and decreases the number of M1 macrophages in the ischemic brain of T1DM-stroke rats. In vitro, VT treatment significantly decreases endothelial cell death and decreases MCP-1, endothelin-1, and VEGF expression under high glucose (HG) and ischemic conditions and significantly increases capillary tube formation under HG conditions when compared to non-treated control group. VT treatment significantly decreases inflammatory factor expression such as MMP9 and MCP-1 in macrophages subjected to LPS activation and significantly decreases IL-1ß and MMP9 expression in PCN subjected to ischemia under HG conditions. CONCLUSION: Delayed VT treatment (24 h after stroke) significantly improves neurological function, promotes vascular and white matter remodeling, and decreases inflammation in the ischemic brain after stroke in T1DM rats.

HUCBC Treatment Improves Cognitive Outcome in Rats With Vascular Dementia.

Background and purpose: Vascular dementia (VaD) is the second common cause of dementia after Alzheimer's disease in older people. Yet, there are no FDA approved drugs specifically for VaD. In this study, we have investigated the therapeutic effects of human umbilical cord blood cells (HUCBC) treatment on the cognitive outcome, white matter (WM) integrity, and glymphatic system function in rats subject to a multiple microinfarction (MMI) model of VaD. Methods: Male, retired breeder rats were subjected to the MMI model (800 ± 100 cholesterol crystals/300 µl injected into the internal carotid artery), and 3 days later were treated with phosphate-buffered saline (PBS) or HUCBC (5 × 106, i.v.). Sham rats were included as naïve control. Following a battery of cognitive tests, rats were sacrificed at 28 days after MMI and brains extracted for immunohistochemical evaluation and Western blot analysis. To evaluate the glymphatic function, fluorescent tracers (Texas Red dextran, MW: 3 kD and FITC-dextran, MW: 500 kD) was injected into the cisterna magna over 30 min at 14 days after MMI. Rats (3-4/group/time point) were sacrificed at 30 min, 3 h, and 6 h, and the tracer movement analyzed using laser scanning confocal microscopy. Results: Compared to control MMI rats, HUCBC treated MMI rats exhibit significantly improved short-term memory and long-term memory exhibited by increased discrimination index in novel object recognition task with retention delay of 4 h and improved novel odor recognition task with retention delay of 24 h, respectively. HUCBC treatment also improves spatial learning and memory as measured using the Morris water maze test compared to control MMI rats. HUCBC treatment significantly increases axon and myelin density increases oligodendrocyte and oligodendrocyte progenitor cell number and increases Synaptophysin expression in the brain compared to control MMI rats. HUCBC treatment of MMI in rats significantly improves glymphatic function by reversing MMI induced delay in the penetration of cerebrospinal fluid (CSF) into the brain parenchyma via glymphatic pathways and reversing delayed clearance from the brain. HUCBC treatment significantly increases miR-126 expression in serum, aquaporin-4 (AQP4) expression around cerebral vessels, and decreases transforming growth factor-ß (TGF-ß) protein expression in the brain which may contribute to HUCBC induced improved glymphatic function. Conclusions: HUCBC treatment of an MMI rat model of VaD promotes WM remodeling and improves glymphatic function which together may aid in the improvement of cognitive function and memory. Thus, HUCBC treatment warrants further investigation as a potential therapy for VaD.

Exosomes derived from bone marrow mesenchymal stem cells harvested from type two diabetes rats promotes neurorestorative effects after stroke in type two diabetes rats.

BACKGROUND AND PURPOSE: Diabetes elevates the risk of stroke, promotes inflammation, and exacerbates vascular and white matter damage post stroke, thereby hindering long term functional recovery. Here, we investigated the neurorestorative effects and the underlying therapeutic mechanisms of treatment of stroke in type 2 diabetic rats (T2DM) using exosomes harvested from bone marrow stromal cells obtained from T2DM rats (T2DM-MSC-Exo). METHODS: T2DM was induced in adult male Wistar rats using a combination of high fat diet and Streptozotocin. Rats were subjected to transient 2 h middle cerebral artery occlusion (MCAo) and 3 days later randomized to one of the following treatment groups: 1) phosphate-buffered-saline (PBS, i.v), 2) T2DM-MSC-Exo, (3 × 1011, i.v), 3) T2DM-MSC-Exo with miR-9 over expression (miR9+/+-T2DM-MSC-Exo, 3 × 1011, i.v) or 4) MSC-Exo derived from normoglycemic rats (Nor-MSC-Exo) (3 × 1011, i.v). T2DM sham control group is included as reference. Rats were sacrificed 28 days after MCAo. RESULTS: T2DM-MSC-Exo treatment does not alter blood glucose, lipid levels, or lesion volume, but significantly improves neurological function and attenuates post-stroke weight loss compared to PBS treated as well as Nor-MSC-Exo treated T2DM-stroke rats. Compared to PBS treatment, T2DM-MSC-Exo treatment of T2DM-stroke rats significantly 1) increases tight junction protein ZO-1 and improves blood brain barrier (BBB) integrity; 2) promotes white matter remodeling indicated by increased axon and myelin density, and increases oligodendrocytes and oligodendrocyte progenitor cell numbers in the ischemic border zone as well as increases primary cortical neuronal axonal outgrowth; 3) decreases activated microglia, M1 macrophages, and inflammatory factors MMP-9 (matrix mettaloproteinase-9) and MCP-1 (monocyte chemoattractant protein-1) expression in the ischemic brain; and 4) decreases miR-9 expression in serum, and increases miR-9 target ABCA1 (ATP-binding cassette transporter 1) and IGFR1 (Insulin-like growth factor 1 receptor) expression in the brain. MiR9+/+-T2DM-MSC-Exo treatment significantly increases serum miR-9 expression compared to PBS treated and T2DM-MSC-Exo treated T2DM stroke rats. Treatment of T2DM stroke with miR9+/+-T2DM-MSC-Exo fails to improve functional outcome and attenuates T2DM-MSC-Exo treatment induced white matter remodeling and anti-inflammatory effects in T2DM stroke rats. CONCLUSIONS: T2DM-MSC-Exo treatment for stroke in T2DM rats promotes neurorestorative effects and improves functional outcome. Down regulation of miR-9 expression and increasing its target ABCA1 pathway may contribute partially to T2DM-MSC-Exo treatment induced white matter remodeling and anti-inflammatory responses.

ABCA1/ApoE/HDL Signaling Pathway Facilitates Myelination and Oligodendrogenesis after Stroke.

ATP-binding cassette transporter A1 (ABCA1) plays an important role in the regulation of apolipoprotein E (ApoE) and the biogenesis of high-density lipoprotein (HDL) cholesterol in the mammalian brain. Cholesterol is a major source for myelination. Here, we investigate whether ABCA1/ApoE/HDL contribute to myelin repair and oligodendrogenesis in the ischemic brain after stroke. Specific brain ABCA1-deficient (ABCA1-B/-B) and ABCA1-floxed (ABCA1fl/fl) control mice were subjected to permanent distal middle-cerebral-artery occlusion (dMCAo) and were intracerebrally administered (1) artificial mouse cerebrospinal fluid (CSF) as vehicle control, (2) human plasma HDL3, and (3) recombined human ApoE2 starting 24 h after dMCAo for 14 days. All stroke mice were sacrificed 21 days after dMCAo. The ABCA1-B/-B-dMCAo mice exhibit significantly reduced myelination and oligodendrogenesis in the ischemic brain as well as decreased functional outcome 21 days after stroke compared with ABCA1fl/fl mice; administration of human ApoE2 or HDL3 in the ischemic brain significantly attenuates the deficits in myelination and oligodendrogenesis in ABCA1-B/-B-dMCAo mice ( p < 0.05, n = 9/group). In vitro, ABCA1-B/-B reduces ApoE expression and decreases primary oligodendrocyte progenitor cell (OPC) migration and oligodendrocyte maturation; HDL3 and ApoE2 treatment significantly reverses ABCA1-B/-B-induced reduction in OPC migration and oligodendrocyte maturation. Our data indicate that the ABCA1/ApoE/HDL signaling pathway contributes to myelination and oligodendrogenesis in the ischemic brain after stroke.

Spleen associated immune-response mediates brain-heart interaction after intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Intracerebral hemorrhage (ICH) patients frequently encounter cardiovascular complications which may contribute to increased mortality and poor long term outcome. ICH induces systemic oxidative stress and activates peripheral immune responses which are involved in the pathological cascade leading to cardiac dysfunction and heart failure after ICH. We have previously reported that ICH induces progressive cardiac dysfunction in mice without primary cardiac diseases. In this study, we have investigated the role of immune response in mediating cardiac dysfunction post ICH in mice. METHODS: Adult male C57BL/6 J mice were randomly assigned to the following groups (n = 8/group): 1) sham control; 2) ICH; 3) splenectomy with ICH (ICH + Spx); 4) splenectomy alone (Spx). Echocardiography was performed at 7 and 28 days after ICH. A battery of neurological and cognitive tests were performed. Flow cytometry, western blot and immunostaining were used to test mechanisms of ICH induced cardiac dysfunction. RESULTS: Compared to sham control mice, Spx alone does not induce acute (7 day) or chronic (28 day) cardiac dysfunction. ICH induces significant neurological and cognitive deficits, as well as acute and chronic cardiac dysfunction compared to sham control mice. Mice subjected to ICH + Spx exhibit significantly improved neurological and cognitive function compared to ICH mice. Mice with ICH + Spx also exhibit significantly improved acute and chronic cardiac function compared to ICH mice indicated by increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), decreased cardiac fibrosis, decreased cardiomyocyte hypertrophy, decreased cardiac infiltration of immune cells and decreased expression of inflammatory factor and oxidative stress in the heart. CONCLUSIONS: Our study demonstrates that splenectomy attenuates ICH-induced neurological and cognitive impairment as well as ICH-induced cardiac dysfunction in mice. Inflammatory cell infiltration into heart and immune responses mediated by the spleen may contribute to ICH-induce acute and chronic cardiac dysfunction and pathological cardiac remodeling.

Inflammatory responses mediate brain-heart interaction after ischemic stroke in adult mice.

Stroke induces cardiac dysfunction which increases post stroke mortality and morbidity particularly in aging population. Here, we investigated the effects of inflammatory responses as underlying mediators of cardiac dysfunction after stroke in adult mice. Adult (eight-to-nine months) male C57BL/6 mice were subjected to photothrombotic stroke. To test whether immunoresponse to stroke leads to cardiac dysfunction, splenectomy was performed with stroke. Immunohistochemistry, flow cytometry, PCR, ELISA and echocardiography were performed. We found marginal cardiac dysfunction at acute phase and significant cardiac dysfunction at chronic phase of stroke as indicated by significant decrease of left ventricular ejection fraction (LVEF) and shortening fraction (LVSF). Stroke significantly increases macrophage infiltration into the heart and increases IL-1ß, IL-6, MCP-1, TGF-ß and macrophage-associated inflammatory cytokine levels in the heart as well as induces cardiac-fibrosis and hypertrophy. Splenectomy with stroke significantly reduces macrophage infiltration into heart, decreases inflammatory factor expression in the heart, decreases cardiac hypertrophy and fibrosis, as well as significantly improves cardiac function compared to non-splenectomized adult stroke mice. Therefore, cerebral ischemic stroke in adult mice induces chronic cardiac dysfunction and secondary immune response may contribute to post stroke cardiac dysfunction.

ApoA-I Mimetic Peptide Reduces Vascular and White Matter Damage After Stroke in Type-2 Diabetic Mice.

Diabetes leads to an elevated risk of stroke and worse functional outcome compared to the general population. We investigate whether L-4F, an economical ApoA-I mimetic peptide, reduces neurovascular and white-matter damage in db/db type-2 diabetic (T2DM) stroke mice. L-4F (16 mg/kg, subcutaneously administered initially 2 h after stroke and subsequently daily for 4 days) reduced hemorrhagic transformation, decreased infarct-volume and mortality, and treated mice exhibited increased cerebral arteriole diameter and smooth muscle cell number, decreased blood-brain barrier leakage and white-matter damage in the ischemic brain as well as improved neurological functional outcome after stroke compared with vehicle-control T2DM mice (p < 0.05, n = 11/group). Moreover, administration of L-4F mitigated macrophage infiltration, and reduced the level of proinflammatory mediators tumor necrosis factor alpha (TNFα), high-mobility group box-1 (HMGB-1)/advanced glycation end-product receptor (RAGE) and plasminogen activator inhibitor-1 (PAI-1) in the ischemic brain in T2DM mice (p < 0.05, n = 6/group). In vitro, L-4F treatment did not increase capillary-like tube formation in mouse-brain endothelial cells, but increased primary artery explant cell migration derived from C57BL/6-aorta 1 day after middle cerebral artery occlusion (MCAo), and enhanced neurite-outgrowth after 2 h of oxygen-glucose deprivation and axonal-outgrowth in primary cortical neurons derived from the C57BL/6-embryos subjected to high-glucose condition. This study suggests that early treatment with L-4F provides a potential strategy to reduce neuroinflammation and vascular and white-matter damage in the T2DM stroke population.

MiR-126 Mediates Brain Endothelial Cell Exosome Treatment-Induced Neurorestorative Effects After Stroke in Type 2 Diabetes Mellitus Mice.

Background and Purpose- Stroke patients with type 2 diabetes mellitus (T2DM) exhibit increased vascular and white matter damage and have worse prognosis compared with nondiabetic stroke patients. We investigated the neurorestorative effects of exosomes derived from mouse brain endothelial cells (EC-Exo) as treatment for stroke in T2DM mice and investigated the role of miR-126 in mediating EC-Exo-derived therapeutic benefits in T2DM-stroke mice. Methods- Adult, male BKS.Cg-m+/+Leprdb/J (T2DM) mice were subjected to photothrombotic stroke model. T2DM mice were intravenously injected at 3 days after stroke with (1) PBS; (2) liposome mimic (vehicle control, 3×1010); (3) EC-Exo (3×1010); (4) knockdown of miR-126 in EC-Exo (miR-126-/- EC-Exo, 3×1010). Behavioral and cognitive tests were performed, and mice were sacrificed at 28 days after stroke. Results- Compared with non-DM stroke mice, T2DM-stroke mice exhibit significantly decreased serum and brain tissue miR-126 expression. Endothelial cells and EC-Exo contain high levels of miR-126 compared with other cell types or exosomes derived from other types of cells, respectively (smooth muscle cells, astrocytes, and marrow stromal cells). Compared with PBS or liposome mimic treatment, EC-Exo treatment of T2DM-stroke mice significantly improves neurological and cognitive function, increases axon density, myelin density, vascular density, arterial diameter, as well as induces M2 macrophage polarization in the ischemic boundary zone. MiR-126-/- EC-Exo treatment significantly decreases miR-126 expression in serum and brain, as well as attentuates EC-Exo treatment-induced functional improvement and does not significantly increase axon and myelin density, vascular density, arterial diameter or induce M2 macrophage polarization in T2DM-stroke mice. In vitro, EC-Exo treatment significantly increases primary cortical neuron axonal outgrowth and increases endothelial capillary tube formation whereas miR-126-/- EC-Exo attentuates EC-Exo induced capillary tube formation and axonal outgrowth. Conclusions- EC-Exo treatment of stroke promotes neurorestorative effects in T2DM mice. MiR-126 may mediate EC-Exo-induced neurorestorative effects in T2DM mice. Visual Overview- An online visual overview is available for this article.

Deficiency of tPA Exacerbates White Matter Damage, Neuroinflammation, Glymphatic Dysfunction and Cognitive Dysfunction in Aging Mice.

Tissue plasminogen activator (tPA) is a serine protease primarily involved in mediating thrombus breakdown and regulating catabolism of amyloid-beta (Aß). The aim of this study is to investigate age-dependent decline of endogenous tPA and the effects of tPA decline on glymphatic function and cognitive outcome in mice. Male, young (3m), adult (6m) and middle-aged (12m) C57/BL6 (wild type) and tPA knockout (tPA-/-) mice were subject to a battery of cognitive tests and white matter (WM) integrity, neuroinflammation, and glymphatic function were evaluated. Adult WT mice exhibit significantly decreased brain tPA level compared to young WT mice and middle-aged WT mice have significantly lower brain tPA levels than young and adult WT mice. Middle-aged WT mice exhibit significant neuroinflammation, reduced WM integrity and increased thrombin deposition compared to young and adult mice, and increased blood brain barrier (BBB) permeability and reduced cognitive ability compared to young WT mice. In comparison to adult WT mice, adult tPA-/- mice exhibit significant BBB leakage, decreased dendritic spine density, increased thrombin deposition, neuroinflammation, and impaired functioning of the glymphatic system. Compared to age-matched WT mice, adult and middle-aged tPA-/- mice exhibit significantly increased D-Dimer expression and decreased perivascular Aquaporin-4 expression. Compared to age-matched WT mice, young, adult and middle-aged tPA-/- mice exhibit significant cognitive impairment, axonal damage, and increased deposition of amyloid precursor protein (APP), Aß, and fibrin. Endogenous tPA may play an important role in contributing to aging induced cognitive decline, axonal/WM damage, BBB disruption and glymphatic dysfunction in the brain.

Sildenafil treatment of vascular dementia in aged rats.

BACKGROUND: and purpose: In this study, we employed a multiple microinfarction (MMI) based vascular dementia (VaD) model in aged rats and tested the therapeutic effects of Sildenafil, a phosphodiesterase type 5 inhibitor, on cognitive decline, white matter damage, autophagy and inflammatory response associated with VaD. METHODS: Male, aged (16-18 months) Wistar rats were subjected to MMI (800 ± 100, 70-100 µm cholesterol crystals injected into the internal carotid artery) and treated with or without Sildenafil (2 mg/kg, i.p) starting at 24 h after MMI daily for 28 days. Four experimental groups were employed: Sham control, Sham + Sildenafil, MMI, and MMI + Sildenafil. A battery of cognitive tests were performed and rats were sacrificed at 28 days after MMI for immunohistochemical evaluation and PCR assay. RESULTS: Sildenafil treatment in aged MMI rats significantly improves short term memory evaluated by the novel object recognition test and improves spatial learning and memory in the Morris water maze test compared to aged control MMI rats. Sildenafil treatment of aged MMI rats significantly increases axon and myelin density in the corpus callosum and white matter bundles in the striatum, increases oligodendrocyte and oligodendrocyte progenitor cell number in the corpus callosum, cortex and striatum, and increases synaptic protein expression in the cortex and striatum compared to aged control MMI rats. In addition, Sildenafil treatment of MMI in aged rats significantly decreases Beclin1 expression and inflammatory factors Monocyte chemoattractant protein-1 and Interleukin-1ß expression in brain. Sildenafil treatment in aged rats does not improve cognitive outcome compared to aged sham control rats. CONCLUSIONS: Sildenafil treatment of MMI in aged rats significantly improves cognition and memory at 1 month after MMI. Sildenafil treatment increases axon and myelin density, increases Synaptophysin expression, decreases autophagic activity and exerts anti-inflammatory effects which in concert may contribute to cognitive improvement in aged rats subjected to MMI.