Microvascular Network Remodeling in the Ischemic Mouse Brain Defined by Light Sheet Microscopy.

BACKGROUND: Until now, the analysis of microvascular networks in the reperfused ischemic brain has been limited due to tissue transparency challenges. METHODS: Using light sheet microscopy, we assessed microvascular network remodeling in the striatum from 3 hours to 56 days post-ischemia in 2 mouse models of transient middle cerebral artery occlusion lasting 20 or 40 minutes, resulting in mild ischemic brain injury or brain infarction, respectively. We also examined the effect of a clinically applicable S1P (sphingosine-1-phosphate) analog, FTY720 (fingolimod), on microvascular network remodeling. RESULTS: Over 56 days, we observed progressive microvascular degeneration in the reperfused striatum, that is, the lesion core, which was followed by robust angiogenesis after mild ischemic injury induced by 20-minute middle cerebral artery occlusion. However, more severe ischemic injury elicited by 40-minute middle cerebral artery occlusion resulted in incomplete microvascular remodeling. In both cases, microvascular networks did not return to their preischemic state but displayed a chronically altered pattern characterized by higher branching point density, shorter branches, higher unconnected branch density, and lower tortuosity, indicating enhanced network connectivity. FTY720 effectively increased microvascular length density, branching point density, and volume density in both models, indicating an angiogenic effect of this drug. CONCLUSIONS: Utilizing light sheet microscopy together with automated image analysis, we characterized microvascular remodeling in the ischemic lesion core in unprecedented detail. This technology will significantly advance our understanding of microvascular restorative processes and pave the way for novel treatment developments in the stroke field.

Neural precursor cell delivery induces acute post-ischemic cerebroprotection, but fails to promote long-term stroke recovery in hyperlipidemic mice due to mechanisms that include pro-inflammatory responses associated with brain hemorrhages.

BACKGROUND: The intravenous delivery of adult neural precursor cells (NPC) has shown promising results in enabling cerebroprotection, brain tissue remodeling, and neurological recovery in young, healthy stroke mice. However, the translation of cell-based therapies to clinical settings has encountered challenges. It remained unclear if adult NPCs could induce brain tissue remodeling and recovery in mice with hyperlipidemia, a prevalent vascular risk factor in stroke patients. METHODS: Male mice on a normal (regular) diet or on cholesterol-rich Western diet were exposed to 30 min intraluminal middle cerebral artery occlusion (MCAO). Vehicle or 106 NPCs were intravenously administered immediately after reperfusion, at 3 day and 7 day post-MCAO. Neurological recovery was evaluated using the Clark score, Rotarod and tight rope tests over up to 56 days. Histochemistry and light sheet microscopy were used to examine ischemic injury and brain tissue remodeling. Immunological responses in peripheral blood and brain were analyzed through flow cytometry. RESULTS: NPC administration reduced infarct volume, blood-brain barrier permeability and the brain infiltration of neutrophils, monocytes, T cells and NK cells in the acute stroke phase in both normolipidemic and hyperlipidemic mice, but increased brain hemorrhage formation and neutrophil, monocyte and CD4+ and CD8+ T cell counts and activation in the blood of hyperlipidemic mice. While neurological deficits in hyperlipidemic mice were reduced by NPCs at 3 day post-MCAO, NPCs did not improve neurological deficits at later timepoints. Besides, NPCs did not influence microglia/macrophage abundance and activation (assessed by morphology analysis), astroglial scar formation, microvascular length or branching point density (evaluated using light sheet microscopy), long-term neuronal survival or brain atrophy in hyperlipidemic mice. CONCLUSIONS: Intravenously administered NPCs did not have persistent effects on post-ischemic neurological recovery and brain remodeling in hyperlipidemic mice. These findings highlight the necessity of rigorous investigations in vascular risk factor models to fully assess the long-term restorative effects of cell-based therapies. Without comprehensive studies in such models, the clinical potential of cell-based therapies cannot be definitely determined.

Rapid and fully automated blood vasculature analysis in 3D light-sheet image volumes of different organs.

Light-sheet fluorescence microscopy (LSFM) can produce high-resolution tomograms of tissue vasculature with high accuracy. However, data processing and analysis is laborious due to the size of the datasets. Here, we introduce VesselExpress, an automated software that reliably analyzes six characteristic vascular network parameters including vessel diameter in LSFM data on average computing hardware. VesselExpress is ∼100 times faster than other existing vessel analysis tools, requires no user interaction, and integrates batch processing and parallelization. Employing an innovative dual Frangi filter approach, we show that obesity induces a large-scale modulation of brain vasculature in mice and that seven other major organs differ strongly in their 3D vascular makeup. Hence, VesselExpress transforms LSFM from an observational to an analytical working tool.

Small extracellular vesicles obtained from hypoxic mesenchymal stromal cells have unique characteristics that promote cerebral angiogenesis, brain remodeling and neurological recovery after focal cerebral ischemia in mice.

Obtained from the right cell-type, mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) promote stroke recovery. Within this process, microvascular remodeling plays a central role. Herein, we evaluated the effects of MSC-sEVs on the proliferation, migration, and tube formation of human cerebral microvascular endothelial cells (hCMEC/D3) in vitro and on post-ischemic angiogenesis, brain remodeling and neurological recovery after middle cerebral artery occlusion (MCAO) in mice. In vitro, sEVs obtained from hypoxic (1% O2), but not 'normoxic' (21% O2) MSCs dose-dependently promoted endothelial proliferation, migration, and tube formation and increased post-ischemic endothelial survival. sEVs from hypoxic MSCs regulated a distinct set of miRNAs in hCMEC/D3 cells previously linked to angiogenesis, three being upregulated (miR-126-3p, miR-140-5p, let-7c-5p) and three downregulated (miR-186-5p, miR-370-3p, miR-409-3p). LC/MS-MS revealed 52 proteins differentially abundant in sEVs from hypoxic and 'normoxic' MSCs. 19 proteins were enriched (among them proteins involved in extracellular matrix-receptor interaction, focal adhesion, leukocyte transendothelial migration, protein digestion, and absorption), and 33 proteins reduced (among them proteins associated with metabolic pathways, extracellular matrix-receptor interaction, focal adhesion, and actin cytoskeleton) in hypoxic MSC-sEVs. Post-MCAO, sEVs from hypoxic MSCs increased microvascular length and branching point density in previously ischemic tissue assessed by 3D light sheet microscopy over up to 56 days, reduced delayed neuronal degeneration and brain atrophy, and enhanced neurological recovery. sEV-induced angiogenesis in vivo depended on the presence of polymorphonuclear neutrophils. In neutrophil-depleted mice, MSC-sEVs did not influence microvascular remodeling. sEVs from hypoxic MSCs have distinct angiogenic properties. Hypoxic preconditioning enhances the restorative effects of MSC-sEVs.

Dose-Dependent Microglial and Astrocytic Responses Associated With Post-ischemic Neuroprotection After Lipopolysaccharide-Induced Sepsis-Like State in Mice.

In contrast to lipopolysaccharide (LPS)-induced preconditioning, which has repeatedly been examined in the past, the effects of post-ischemic LPS-induced sepsis, although clinically considerably more important, have not systemically been studied. We exposed mice to transient intraluminal middle cerebral artery occlusion (MCAO) and examined the effects of intraperitoneal LPS (0.1 or 1 mg/kg) which was administered 24 h post-ischemia. Post-ischemic glial reactivity, neuronal survival and neurological outcome were differently modulated by the higher and the lower LPS dose. Although both doses promoted neuronal survival after 72 h, the underlying mechanisms were not similar. Mice receiving 1 mg/kg LPS exhibited transient hypothermia at 1 and 3 hours post sepsis (hps), followed by reduced focal neurological deficits at 24, 48 and 72 hps. The lower dose (0.1 mg/kg) did not induce hypothermia, but reduced microglia/macrophage activation with the appearance of an anti-inflammatory CD206 positive cell phenotype in the brain parenchyma. Together, our results indicate a novel, dose-dependent modulation of microglial cells that is intricately involved in brain protection.

Light Sheet Microscopy Using FITC-Albumin Followed by Immunohistochemistry of the Same Rehydrated Brains Reveals Ischemic Brain Injury and Early Microvascular Remodeling.

Until recently, the visualization of cerebral microvessels was hampered by the fact that only short segments of vessels could be evaluated in brain sections by histochemistry. These limitations have been overcome by light sheet microscopy, which allows the 3D analysis of microvasculature in cleared brains. A major limitation of light sheet microscopy is that antibodies do not sufficiently penetrate cleared brains. We herein describe a technique of reverse clearing and rehydration, which after microvascular network analysis allows brain sectioning and immunohistochemistry employing a broad set of antibodies. Performing light sheet microscopy on brains of mice exposed to intraluminal middle cerebral artery occlusion (MCAO), we show that in the early phase of microvascular remodeling branching point density was markedly reduced, more strongly than microvascular length. Brain infarcts in light sheet microscopy were sharply demarcated by their autofluorescence signal, closely corresponding to brain infarcts revealed by Nissl staining. Neuronal survival, leukocyte infiltration, and astrocytic reactivity could be evaluated by immunohistochemistry in rehydrated brains, as shown in direct comparisons with non-cleared brains. Immunohistochemistry revealed microthrombi in ischemic microvessels that were likely responsible for the marked branching point loss. The balance between microvascular thrombosis and remodeling warrants further studies at later time-points after stroke.

Therapeutic Effect Evaluation of Neuromuscular Electrical Stimulation With or Without Strengthening Exercise on Spastic Cerebral Palsy.

The aims of this study were to investigate the effect of neuromuscular electrical stimulation (NMES) combined with strengthening exercise on movement in children with spastic cerebral palsy (CP). One hundred children with spastic CP were randomly divided into a treatment group (NMES and strengthening exercise, n = 50) and a control group (only NMES, n = 50). We compared the Comprehensive Spasticity Scale (CSS) score, Gross Motor Function Measure (GMFM) score, and walking speed before treatment and 6 weeks and 3 months after treatment between the 2 groups. There was no difference in CSS score between the treatment and control groups before the therapy (12.0 ± 3.4 vs 12.3 ± 3.6), which decreased much more in the treatment group after 6 weeks (7.6 ± 3.0 vs 9.5 ± 2.8) and 3 months (7.4 ± 2.4 vs 9.4 ± 2.6) with significant differences ( P < .05). No difference in GMFM score was observed between the treatment and control groups before the therapy (44.5 ± 13.2 vs 44.0 ± 12.6), which increased much more in the treatment group after 6 weeks (70.6 ± 15.2 vs 56.7 ± 14.3) and 3 months (71.0 ± 16.4 vs 58.0 ± 15.6) with significant differences ( P < .05). The walking speed improved over time, which was the same before the treatment (0.43 ± 0.13 m/s vs 0.45 ± 0.14 m/s), and was significantly greater in the treatment group than that in the control group (6 weeks: 0.69 ± 0.15 m/s vs 0.56 ± 0.12 m/s, P < .05; 3 months: 0.72 ± 0.17 m/s vs 0.57 ± 0.18 m/s, P < .05). NMES combined with strengthening exercise was more effective than NMES alone in the recovery of spastic CP.

Taxane-derived compounds protect SK-N-SH cells against oxidative stress injury induced by H2O2.

OBJECTIVE: Discussed the protection of taxane-derived compounds, 7-deacetyl-taxine B and 5-cinnamoyloxy-taxin B, against oxidative stress injury. METHODS: SK-N-SH cells were pretreated with 7-deacetyl-taxine B, 5-cinnamoyloxy-taxin B or DMSO (control) and then incubated with H2O2 for another 24 h. Cell viability was measured by MTT colorimetric assay. Apoptosis rate, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected using flow cytometry assay. Ultrastructure was detected by Transmission electron microscope. RESULTS: Taxane-derived compounds improved H2O2 mediated loss of cell viability in SK-N-SH cell. Ultrastructure of control group cells showed nucleus shrinkage, dense aggregation of chromatin, appearance of apoptotic body and mitochondrial dilation. Treated group showed alleviation of mitochondrial dilation and chromatin margination, and reduction of apoptosis cells induced by H2O2. The apoptosis rate was decreased in treated group compared with control group. DCFH-DA staining showed that ROS were significantly decreased in cells treated with taxane-derived compounds compared with control group. Rhodamine123 staining showed that MMP was significantly decreased in cells treated with taxane-derived compounds compared with control group. CONCLUSION: The anti-oxidative stress and neuroprotective effect of taxane-derived compounds was verified here, wherein it could protect neurocytes from H2O2-induced oxidative stress injury.

Effect of acupuncture on inflammatory cytokines expression of spastic cerebral palsy rats.

OBJECTIVE: To To investigate the effect of acupuncture on the tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP), nitric oxide synthase (NOS) content and muscular tension of spasticity cerebral palsy rat model. METHODS: The rats with spastic cerebral palsy were randomly divided into the control group, model group and acupuncture group. After successful modeling, the muscular tension and the content of TNF-α, IL-6, CRP, NOS were measured. RESULTS: The serum TNF-α, IL-6, CRP, NOS content were significantly decreased in the acupuncture group (P<0.05). The low and high shear viscosity of whole blood of the acupuncture group were significantly lower than the control group and the model group (P<0.05). The erythrocyte electrophoresis indexes in the acupuncture group were significantly lower than that in the model group and the control group (P<0.05). Acupuncture significantly reduced the muscular tension of spastic cerebral palsy rat and increased the active extent in the paralytic extremity (P<0.05), but it could not be restored to normal level. Compared with the control group, the difference had significant (P<0.05). CONCLUSIONS: Acupuncture treatment can inhibit the release of inflammatory cells after brain injury, then reduce immune injury, relieve muscle spasms and reduce muscular tension.

Comparison of earthworm bioaccumulation between readily desorbable and desorption-resistant naphthalene: implications for biouptake routes.

The bioaccumulation of readily desorbable naphthalene and that of desorption-resistant naphthalene in earthworms were compared to examine the effect of desorption resistance on bioavailability of hydrophobic organic contaminants in soil. A series of naphthalene-contaminated soil samples representing different degrees of desorption resistance were prepared using a batch sorption-repetitive desorption approach, and bioaccumulation of these samples was evaluated using earthworms (Eisenia fetida). Soil samples representing high-degree of desorption resistance exhibited considerably lower bioavailability, as indicated by the lower body burden (naphthalene concentration in worm tissue) at a given sorbed-phase naphthalene concentration. Moreover, the body burden of the highly desorption-resistant samples exhibited a significantly different pore-water dependency than that of the readily desorbable samples, indicating that different biouptake mechanisms are likely controlling readily desorbable contaminants and desorption-resistant contaminants. We propose that for readily desorbable contaminants, the primary biouptake route is the pore-water uptake, but for desorption-resistant contaminants enhanced uptake from ingested soil particles can also be important. The surfactant-like substances in worm gut fluids and physical stress such as abrasion are the likely causes for the enhanced release of desorption-resistant contaminants in worm guts. The difference in bioavailability between readily desorbable and desorption-resistant contaminants needs to be taken into account in risk assessment practices.