Neurovascular protection by post‐ischemic intravenous injections of the lipoxin A4 receptor agonist,BML‐111, in a rat model of ischemic stroke

Resolution of inflammation is an emerging new strategy to reduce damage following ischemic stroke. Lipoxin A₄ (LXA₄) is an anti‐inflammatory, pro‐resolution lipid mediator with high affinity binding to ALX, the lipoxin A₄ receptor. Since LXA₄ is rapidly inactivated, potent analogs have been created, including the ALX agonist BML‐111. We hypothesized that post‐ischemic intravenous administration of BML‐111 would provide protection to the neurovascular unit and reduce neuroinflammation in a rat stroke model. Animals were subjected to 90 min of middle cerebral artery occlusion (MCAO) and BML‐111 was injected 100 min and 24 h after stroke onset and animals euthanized at 48 h. Post‐ischemic treatment with BML‐111 significantly reduced infarct size, decreased vasogenic edema, protected against blood–brain barrier disruption, and reduced hemorrhagic transformation. Matrix metalloproteinase‐9 and matrix metalloproteinase‐3 were significantly reduced following BML‐111 treatment. Administration of BML‐111 dramatically decreased microglial activation, as seen with CD68, and neutrophil infiltration and recruitment, as assessed by levels of myeloperoxidase and intracellular adhesion molecule‐1. The tight junction protein zona occludens‐1 was protected from degradation following treatment with BML‐111. These results indicate that post‐ischemic activation of ALX has pro‐resolution effects that limit the inflammatory damage in the cerebral cortex and helps maintain blood–brain barrier integrity after ischemic stroke.

     

A novel caffeoyl triterpene attenuates cerebral ischemic injury with potent anti‐inflammatory and hypothermic effects

Despite the intense efforts in searching for stroke therapies, an urgent need still exists to explore novel neuroprotective agents for ischemic stroke that have high efficacy and wide therapeutic time‐window. Here, we provide the first demonstration that 28‐O‐caffeoyl betulin (B‐CA), a novel derivative of naturally occurring caffeoyl triterpene, could significantly alleviate brain infarction and neurological deficit when given as late as 6 h after transient middle cerebral artery occlusion in the rat. Moreover, post‐ischemia B‐CA administration exhibited long‐term (14 days post stroke) protective effects on both brain infarction and functional (i.e., motor and sensory) deficits. Protective B‐CA effects correlated with decreased inflammatory responses as indicated by inhibition of microglia and astrocyte activation [stained with ionized calcium‐binding adapter molecule 1 (Iba‐1) and glial fibrillary acidic protein (GFAP) antibody, respectively], as well as suppression of tumor necrosis factor‐α, interleukin‐1β, and cyclooxygenase‐2 overproduction in the ipsilateral cortex of ischemic rat. B‐CA administration caused significant hypothermia in the focal cerebral ischemic rat, which may contribute to its ameliorative effects on brain damage and inflammation. In view of its potency in wide therapeutic time‐window, robust anti‐inflammatory and hypothermic effects, this novel caffeoyl triterpene derivative may lead toward the development of effective therapeutic strategies for the treatment of ischemic stroke.

     

The use of intensity‐based Doppler variance method for single vessel response to functional neurovascular activation

This study presents 1 use of optical coherence tomography (OCT) angiography technique to examine neurovascular coupling effect. Repeated B‐scans OCT recording is performed on the rat somatosensory cortex with cranial window preparation while its contralateral forepaw is electrically stimulated to activate the neurons in rest. We use an intensity‐based Doppler variance (IBDV) algorithm mapped cerebral blood vessels in the cortex, and the temporal alteration in blood perfusion during neurovascular activation is analyzed using the proposed IBDV quantitative parameters. By using principal component analysis‐based Fuzzy C Means clustering method, the stimulus‐evoked vasomotion patterns were classified into 3 categories. We found that the response time of small vessels (resting diameter 14.9 ±6.6 μm), middle vessels (resting diameter 21.1 ±7.9 μm) and large vessels (resting diameter 50.7 ±6.5 μm) to achieve 5% change of vascular dilation after stimulation was 1.5, 2 and 5.5 seconds, respectively. Approximately 5% peak change of relative blood flow (RBF) in both small and middle vessels was observed. The large vessels react slowly and their responses nearly 4 seconds delayed, but no significant change in RBF of the large vessels was seen.      

Hydrogen sulfide protects blood–brain barrier integrity following cerebral ischemia

By using two structurally unrelated hydrogen sulfide (H₂S) donors 5‐(4‐methoxyphenyl) ‐3H‐1, 2‐dithiole‐3‐thione (ADT) and sodium hydrosulfide (NaHS), this study investigated if H₂S protected blood–brain barrier (BBB) integrity following middle cerebral artery occlusion (MCAO). ICR mice underwent MCAO and received H₂S donors at 3 h after reperfusion. Infarction, neurological scores, brain edema, Evans blue (EB) extravasation, and tight junction protein expression were examined at 48 h after MCAO. We also investigated if ADT protected BBB integrity by suppressing post‐ischemic inflammation‐induced Matrix Metalloproteimase‐9 (MMP9) and Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX). ADT increased blood H₂S concentrations, decreased infarction, and improved neurological deficits. Particularly, ADT reduced EB extravasation, brain edema and preserved expression of tight junction proteins in the ischemic brain. NaHS also increased blood H₂S levels and reduced EB extravasation following MCAO. Moreover, ADT inhibited expression of pro‐inflammatory markers induced Nitric Oxide Synthase (iNOS) and IL‐1β while enhanced expression of anti‐inflammatory markers arginase 1 and IL‐10 in the ischemic brain. Accordingly, ADT attenuated ischemia‐induced expression and activity of MMP9. Moreover, ADT reduced NOX‐4 mRNA expression, NOX activity, and inhibited nuclear translocation of Nuclear Factor Kappa‐B (NF‐κB) in the ischemic brain. In conclusion, H₂S donors protected BBB integrity following experimental stroke possibly by acting through NF‐κB inhibition to suppress neuroinflammation induction of MMP9 and NOX4‐derived free radicals.

     

Propensity of <Emphasis Type="Italic">Withania somnifera</Emphasis> to Attenuate Behavioural,Biochemical, and Histological Alterations in Experimental Model of Stroke

The present study was designed to evaluate the beneficial effects of Withania somnifera (WS) pre-supplementation on middle cerebral artery occlusion (MCAO) model of ischemic stroke. Ischemic stroke was induced in the rats by inserting intraluminal suture for 90 min, followed by reperfusion injury for 24 h. The animals were assessed for locomotor functions (by neurological deficit scores, narrow beam walk and rotarod test), cognitive and anxiety-like behavioural functions (by morris water maze and elevated plus maze test). MCAO animals showed significant impairment in locomotor and cognitive functions. Neurobehavioural changes were accompanied by decreased acetylcholinesterase activity, increased oxidative stress in terms of enhanced lipid peroxidation and lowered thiol levels in the MCAO animals. In addition, MCAO animals had cerebral infarcts and the presence of pycnotic nuclei. Single-photon emission computerized tomography (SPECT) of MCAO animals revealed a cerebral infarct as a hypoactive area. On the other hand, pre-supplementation with WS (300 mg/kg body weight) for 30 days to MCAO animals was effective in restoring the acetylcholinesterase activity, lipid peroxidation, thiols and attenuated MCAO induced behavioural deficits. WS significantly reduced the cerebral infarct volume and ameliorated histopathological alterations. Improved blood flow was observed in the SPECT images from the brain regions of ischemic rats pre-treated with WS. The results of the study showed a protective effect of WS supplementation in ischemic stroke and are suggestive of its potential application in stroke management.     

A surgical model of permanent and transient middle cerebral artery stroke in the sheep

Background

Animal models are essential to study the pathophysiological changes associated with focal occlusive stroke and to investigate novel therapies. Currently used rodent models have yielded little clinical success, however large animal models may provide a more suitable alternative to improve clinical translation. We sought to develop a model of acute proximal middle cerebral artery (MCA) ischemic stroke in sheep, including both permanent occlusion and transient occlusion with reperfusion.

Materials and Methods

18 adult male and female Merino sheep were randomly allocated to one of three groups (n = 6/gp): 1) sham surgery; 2) permanent proximal MCA occlusion (MCAO); or 3) temporary MCAO with aneurysm clip. All animals had invasive arterial blood pressure, intracranial pressure and brain tissue oxygen monitoring. At 4 h following vessel occlusion or sham surgery animals were killed by perfusion fixation. Brains were processed for histopathological examination and infarct area determination. 6 further animals were randomized to either permanent (n = 3) or temporary MCAO (n = 3) and then had magnetic resonance imaging (MRI) at 4 h after MCAO.

Results

Evidence of ischemic injury in an MCA distribution was seen in all stroke animals. The ischemic lesion area was significantly larger after permanent (28.8%) compared with temporary MCAO (14.6%). Sham animals demonstrated no evidence of ischemic injury. There was a significant reduction in brain tissue oxygen partial pressure after permanent vessel occlusion between 30 and 210 mins after MCAO. MRI at 4 h demonstrated complete proximal MCA occlusion in the permanent MCAO animals with a diffusion deficit involving the whole right MCA territory, whereas temporary MCAO animals demonstrated MRA evidence of flow within the right MCA and smaller predominantly cortical diffusion deficits.

Conclusions

Proximal MCAO can be achieved in an ovine model of stroke via a surgical approach. Permanent occlusion creates larger infarct volumes, however aneurysm clip application allows for reperfusion.     

Pre‐treatment with the synthetic antioxidant T‐butyl bisphenol protects cerebral tissues from experimental ischemia reperfusion injury

Treatments to inhibit or repair neuronal cell damage sustained during focal ischemia/reperfusion injury in stroke are largely unavailable. We demonstrate that dietary supplementation with the antioxidant di‐tert‐butyl‐bisphenol (BP) before injury decreases infarction and vascular complications in experimental stroke in an animal model. We confirm that BP, a synthetic polyphenol with superior radical‐scavenging activity than vitamin E, crosses the blood–brain barrier and accumulates in rat brain. Supplementation with BP did not affect blood pressure or endogenous vitamin E levels in plasma or cerebral tissue. Pre‐treatment with BP significantly lowered lipid, protein and thiol oxidation and decreased infarct size in animals subjected to middle cerebral artery occlusion (2 h) and reperfusion (24 h) injury. This neuroprotective action was accompanied by down‐regulation of hypoxia inducible factor‐1α and glucose transporter‐1 mRNA levels, maintenance of neuronal tissue ATP concentration and inhibition of pro‐apoptotic factors that together enhanced cerebral tissue viability after injury. That pre‐treatment with BP ameliorates oxidative damage and preserves cerebral tissue during focal ischemic insult indicates that oxidative stress plays at least some causal role in promoting tissue damage in experimental stroke. The data strongly suggest that inhibition of oxidative stress through BP scavenging free radicals in vivo contributes significantly to neuroprotection.

     

Dual‐wavelength hybrid optoacoustic‐ultrasound biomicroscopy for functional imaging of large‐scale cerebral vascular networks

A critical link exists between pathological changes of cerebral vasculature and diseases affecting brain function. Microscopic techniques have played an indispensable role in the study of neurovascular anatomy and functions. Yet, investigations are often hindered by suboptimal trade‐offs between the spatiotemporal resolution, field‐of‐view (FOV) and type of contrast offered by the existing optical microscopy techniques. We present a hybrid dual‐wavelength optoacoustic (OA) biomicroscope capable of rapid transcranial visualization of large‐scale cerebral vascular networks. The system offers 3‐dimensional views of the morphology and oxygenation status of the cerebral vasculature with single capillary resolution and a FOV exceeding 6 × 8 mm², thus covering the entire cortical vasculature in mice. The large‐scale OA imaging capacity is complemented by simultaneously acquired pulse‐echo ultrasound (US) biomicroscopy scans of the mouse skull. The new approach holds great potential to provide better insights into cerebrovascular function and facilitate efficient studies into neurological and vascular abnormalities of the brain.      

Overactivation of NR2B‐containing NMDA receptors through entorhinal–hippocampal connection initiates accumulation of hyperphosphorylated tau in rat hippocampus after transient middle cerebral artery occlusion

Middle cerebral artery occlusion (MCAO) induces secondary damages in the hippocampus that is remote from primary ischemic regions. Tau hyperphosphorylation is an important risk for neurodegenerative diseases. Increased tau phosphorylation has been identified in ischemic cortex, but little is known regarding the changes in the hippocampus. We showed that unilateral transient MCAO induced accumulation of hyperphosphorylated tau and concurrent dephosphorylation of glycogen synthase kinase‐3β at Ser 9 in the ipsilateral hippocampus. These MCAO‐induced changes were not reproduced when glutamatergic inputs from the entorhinal cortex to the hippocampus were transected; however, the changes were mimicked by intrahippocampal N‐methyl‐d ‐aspartate (NMDA) administration. Inhibition of NMDA receptor (NMDAR) subunit NR2B, but not NR2A activity in the hippocampus attenuated the accumulation of hyperphosphorylated tau and spatial cognitive impairment in MCAO rats. Together, our data suggest that overactivation of NR2B‐containing NMDARs through entorhinal–hippocampal connection plays an important role in the accumulation of hyperphosphorylated tau in the hippocampus following MCAO. Glycogen synthase kinase‐3β is an important protein kinase involved in NMDARs‐mediated tau hyperphosphorylation. This study indicates that early inhibition of NR2B‐containing NMDARs may represent a potential strategy to prevent or delay the occurrence of post‐stroke dementia.

     

Functional Real-Time Optoacoustic Imaging of Middle Cerebral Artery Occlusion in Mice

Background and Purpose

Longitudinal functional imaging studies of stroke are key in identifying the disease progression and possible therapeutic interventions. Here we investigate the applicability of real-time functional optoacoustic imaging for monitoring of stroke progression in the whole brain of living animals.

Materials and Methods

The middle cerebral artery occlusion (MCAO) was used to model stroke in mice, which were imaged preoperatively and the occlusion was kept in place for 60 minutes, after which optoacoustic scans were taken at several time points.

Results

Post ischemia an asymmetry of deoxygenated hemoglobin in the brain was observed as a region of hypoxia in the hemisphere affected by the ischemic event. Furthermore, we were able to visualize the penumbra in-vivo as a localized hemodynamically-compromised area adjacent to the region of stroke-induced perfusion deficit.

Conclusion

The intrinsic sensitivity of the new imaging approach to functional blood parameters, in combination with real time operation and high spatial resolution in deep living tissues, may see it become a valuable and unique tool in the development and monitoring of treatments aimed at suspending the spread of an infarct area.     

A simple approach for non‐invasive transcranial optical vascular imaging (nTOVI)

In vivo imaging of cerebral vasculature is highly vital for clinicians and medical researchers alike. For a number of years non‐invasive optical‐based imaging of brain vascular network by using standard fluorescence probes has been considered as impossible. In the current paper controverting this paradigm, we present a robust non‐invasive optical‐based imaging approach that allows visualize major cerebral vessels at the high temporal and spatial resolution. The developed technique is simple to use, utilizes standard fluorescent dyes, inexpensive micro‐imaging and computation procedures. The ability to clearly visualize middle cerebral artery and other major vessels of brain vascular network, as well as the measurements of dynamics of blood flow are presented. The developed imaging approach has a great potential in neuroimaging and can significantly expand the capabilities of preclinical functional studies of brain and notably contribute for analysis of cerebral blood circulation in disorder models.

An example of 1 × 1.5 cm color‐coded image of brain blood vessels of mouse obtained in vivo by transcranial optical vascular imaging (TOVI) approach through the intact cranium.     

Flexible wide‐field optical micro‐angiography based on Fourier‐domain multiplexed dual‐beam swept source optical coherence tomography

Wide‐field optical coherence tomography angiography (OCTA) is gaining interest in clinical imaging applications. In this pursuit, it is challenging to maintain the imaging resolution and sensitivity throughout the wide field of view (FoV). Here, we propose a novel method/system of dual‐beam arrangement and Fourier‐domain multiplexing to achieve wide‐field OCTA when imaging the uneven surface samples. The proposed system provides 2 separate FoVs, with flexibility that the imaging area, focus of the imaging beam and imaging depth range can be individually adjusted for each FoV, leading to either (1) increased system imaging FoV or (2) capability of targeting 2 regions of interests that locate at depths with large difference between each other. We demonstrate this novel method by employing 100 kHz laser source in a swept source OCTA to achieve an effective 200 kHz sweeping rate, covering a 22 × 22 mm FoV. The results are verified by a SS‐OCTA system employing a 200 kHz laser source, together with the experimental demonstrations when imaging whole brain vasculature in rodent models and skin blood perfusion in human fingers, show‐casing the capability of proposed system to image live large samples with complex surface topography.      

MiR‐377 Regulates Inflammation and Angiogenesis in Rats After Cerebral Ischemic Injury

Ischemic stroke is the leading cause of disabilities worldwide. MicroRNA‐377 (miR‐377) plays important roles in ischemic injury. The present study focused on the mechanisms of miR‐377 in protecting ischemic brain injury in rats. Cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. Primary rat microglial cells and brain microvascular endothelial cells (BMECs) were exposed to oxygen‐glucose deprivation (OGD). The concentrations of cytokines (TNF‐α, IL‐1β, IL‐6, IFN‐γ, TGF‐β, MMP2, COX2, and iNOS) in the culture medium were measured by specific ELISA. Tube formation assay was for the in vitro study of angiogenesis. Luciferase reporter assay was performed to confirm whether VEGF and EGR2 were direct targets of miR‐377. The MCAO rats were intracerebroventricular (ICV) injection of miR‐377 inhibitor to assess its protective effects in vivo. MiR‐377 levels were decreased in the rat brain tissues at 1, 3, and 7 d after MCAO. Both microglia cells and BMECs under OGD showed markedly lower expression levels of miR‐377 while higher expression levels of EGR2 and VEGF compared to those under normoxia conditions. Knockdown of miR‐377 inhibited microglial activation and the release of pro‐inflammatory cytokines after OGD. Suppression of miR‐377 promoted the capillary‐like tube formation and cell proliferation and migration of BMECs. The anti‐inflammation effect of EGR2 and the angiogenesis effect of VEGF were regulated by miR‐377 after OGD. Inhibition of miR‐377 decreased cerebral infarct volume and suppressed cerebral inflammation but promoted angiogenesis in MCAO rats. Knockdown of miR‐377 lessened the ischemic brain injury through promoting angiogenesis and suppressing cerebral inflammation. J. Cell. Biochem. 119: 327–337, 2018. © 2017 Wiley Periodicals, Inc.     

The non‐peptidic δ‐opioid receptor agonist Tan‐67 mediates neuroprotection post‐ischemically and is associated with altered amyloid precursor protein expression,maturation and processing in mice

Tan‐67 is a selective non‐peptidic δ‐opioid receptor (DOR ) agonist that confers neuroprotection against cerebral ischemia/reperfusion (I/R)‐caused neuronal injury in pre‐treated animals. In this study, we examined whether post‐ischemic administration of Tan‐67 in stroke mice is also neuroprotective and whether the treatment affects expression, maturation and processing of the amyloid precursor protein (APP ). A focal cerebral I/R model in mice was induced by middle cerebral artery occlusion for 1 h and Tan‐67 (1.5, 3 or 4.5 mg/kg) was administered via the tail vein at 1 h after reperfusion. Alternatively, naltrindole, a selective DOR antagonist (5 mg/kg), was administered 1 h before Tan‐67 treatment. Our results showed that post‐ischemic administration of Tan‐67 (3 mg/kg or 4.5 mg/kg) was neuroprotective as shown by decreased infarct volume and neuronal loss following I/R. Importantly, Tan‐67 improved animal survival and neurobehavioral outcomes. Conversely, naltrindole abolished Tan‐67 neuroprotection in infarct volume. Tan‐67 treatment also increased APP expression, maturation and processing in the ipsilateral penumbral area at 6 h but decreased APP expression and maturation in the same brain area at 24 h after I/R. Tan‐67‐induced increase in APP expression was also seen in the ischemic cortex at 24 h following I/R. Moreover, Tan‐67 attenuated BACE ‐1 expression, β‐secretase activity and the BACE cleavage of APP in the ischemic cortex at 24 h after I/R, which was abolished by naltrindole. Our data suggest that Tan‐67 is a promising DOR ‐dependent therapeutic agent for treating I/R‐caused disorder and that Tan‐67‐mediated neuroprotection may be mediated via modulating APP expression, maturation and processing, despite an uncertain causative relationship between the altered APP and the outcomes observed.

     

MicroRNA‐130a regulates neurological deficit and angiogenesis in rats with ischaemic stroke by targeting XIAP

MicroRNAs (miRNAs) have already been proposed to be implicated in the development of ischaemic stroke. We aim to investigate the role of miR‐130a in the neurological deficit and angiogenesis in rats with ischaemic stroke by regulating X‐linked inhibitor of apoptosis protein (XIAP). Middle cerebral artery occlusion (MCAO) models were established by suture‐occluded method, and MCAO rats were then treated with miR‐130a mimics/inhibitors or/and altered XIAP for detection of changes of rats’ neurological function, nerve damage and angiogenesis in MCAO rats. The oxygen‐glucose deprivation (OGD) cellular models were established and respectively treated to determine the roles of miR‐130a and XIAP in neuronal viability and apoptosis. The expression levels of miR‐130a and XIAP in brain tissues of MCAO rats and OGD‐treated neurons were detected. The binding site between miR‐130a and XIAP was verified by luciferase activity assay. MiR‐130a was overexpressed while XIAP was down‐regulated in MCAO rats and OGD‐treated neurons. In animal models, suppressed miR‐130a improved neurological function, alleviated nerve damage and increased new vessels in brain tissues of rats with MCAO. In cellular models, miR‐130a inhibition promoted neuronal viability and suppressed apoptosis. Inhibited XIAP reversed the effect of inhibited miR‐130a in both MCAO rats and OGD‐treated neurons. XIAP was identified as a target of miR‐130a. Our study reveals that miR‐130a regulates neurological deficit and angiogenesis in rats with MCAO by targeting XIAP.     

Neuroprotective effects of amiodarone in a mouse model of ischemic stroke

Background

Ion channels play a crucial role in the development of ischemic brain injury. Recent studies have reported that the blockade of various types of ion channels improves outcomes in experimental stroke models. Amiodarone, one of the most effective drugs for life-threatening arrhythmia, works as a multiple channel blocker and its characteristics cover all four Vaughan-Williams classes. Although it is known that amiodarone indirectly contributes to preventing ischemic stroke by maintaining sinus rhythm in patients with atrial fibrillation, the direct neuroprotective effect of amiodarone has not been clarified. The purpose of this study was to investigate the direct effect of amiodarone on ischemic stroke in mice.

Methods

Focal cerebral ischemia was induced via distal permanent middle cerebral artery occlusion (MCAO) in adult male mice. The amiodarone pre-treatment group received 50 mg/kg of amiodarone 1 h before MCAO; the amiodarone post-treatment groups received 50 mg/kg of amiodarone immediately after MCAO; the control group received vehicle only. In addition, the sodium channel opener veratrine and selective beta-adrenergic agonist isoprotelenol were used to elucidate the targeted pathway. Heart rate and blood pressure were monitored perioperatively. Infarct volume analysis was conducted 48 h after MCAO. The body asymmetry test and the corner test were used for neurological evaluation.

Results

Amiodarone pre-treatment and post-treatment reduced the heart rate but did not affect the blood pressure. No mice showed arrhythmia. Compared with the control group, the amiodarone pre-treatment group had smaller infarct volumes (8.9?±?2.1% hemisphere [mean?±?SD] vs. 11.2?±?1.4%; P?<?0.05) and improved functional outcomes: lower asymmetric body swing rates (52?±?17% vs. 65?±?18%; P?<?0.05) and fewer left turns (7.1?±?1.2 vs. 8.3?±?1.2; P?<?0.05). In contrast, amiodarone post-treatment did not improve the outcomes after MCAO. The neuroprotective effect of amiodarone pre-treatment was abolished by co-administration of veratrine but not by isoproterenol.

Conclusions

Amiodarone pre-treatment attenuated ischemic brain injury and improved functional outcomes without affecting heart rhythm and blood pressure. The present results showed that amiodarone pre-treatment has neuroprotective effects, at least in part, via blocking the sodium channels.

     

Caveolin‐1 mediates tissue plasminogen activator‐induced MMP‐9 up‐regulation in cultured brain microvascular endothelial cells

Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase‐9 (MMP‐9) activity in the ischemic brain, which exacerbates blood‐brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP‐9 activity is not well understood. Here we report an important role of caveolin‐1 in mediating tPA‐induced MMP‐9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP‐9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP‐9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3‐fold increase of caveolin‐1 protein levels in endothelial cells. Interestingly, knockdown of Cav‐1 with siRNA inhibited tPA‐induced MMP‐9 mRNA up‐regulation and MMP‐9 increase in the conditioned media, but did not affect MMP‐9 decrease in cellular extracts. These results suggest that caveolin‐1 critically contributes to tPA‐mediated MMP‐9 up‐regulation, but may not facilitate MMP‐9 secretion in endothelial cells.

     

High‐throughput light sheet tomography platform for automated fast imaging of whole mouse brain

Acquiring information of the neural structures in the whole‐brain level is vital for systematically exploring mechanisms and principles of brain function and dysfunction. Most methods for whole brain imaging, while capable of capturing the complete morphology of neurons, usually involve complex sample preparation and several days of image acquisition. The whole process including optical clearing or resin embedding is time consuming for a quick survey of the distribution of specific neural circuits in the whole brain. Here, we develop a high‐throughput light‐sheet tomography platform (HLTP), which requires minimum sample preparation. This method does not require optical clearing for block face light sheet imaging. After fixation using paraformaldehyde, an aligned 3 dimensional image dataset of a whole mouse brain can be obtained within 5 hours at a voxel size of 1.30 × 1.30 × 0.92 μm. HLTP could be a very efficient tool for quick exploration and visualization of brain‐wide distribution of specific neurons or neural circuits.