Neurovascular Recovery Research Articles

Abstracts from The 39th Annual Symposium of the National Neurotrauma Society, including the AANS/CNS Joint Section on Neurotrauma and Critical Care

Abstracts from The 39^th Annual Symposium of the National Neurotrauma Society, including the AANS/CNS Joint Section on Neurotrauma and Critical Care

Longitudinal monitoring of microglial/macrophage activation in ischemic rat brain using Iba-1-specific nanoparticle-enhanced magnetic resonance imaging

Microglial/macrophage activation plays a dual role in response to brain injury after a stroke, promoting early neuroinflammation and benefit for neurovascular recovery. Therefore, the dynamics of stroke-induced cerebral microglial/macrophage activation are of substantial interest. This study used novel anti-Iba-1-targeted superparamagnetic iron–platinum (FePt) nanoparticles in conjunction with magnetic resonance imaging (MRI) to measure the spatiotemporal changes of the microglial/macrophage activation in living rat brain for four weeks post-stroke. Ischemic lesion areas were identified and measured using T2-weighted MR images. After injection of the FePt-nanoparticles, T2*-weighted MR images showed that the nanoparticles were seen solely in brain regions that coincided with areas of active microglia/macrophages detected by post-mortem immunohistochemistry. Good agreement in morphological and distributive dynamic changes was also observed between the Fe+-cells and the Iba-1+-microglia/macrophages. The spatiotemporal changes of nanoparticle detected by T2*-weighted images paralleled the changes of microglial/macrophage activation and phenotypes measured by post-mortem immunohistochemistry over the four weeks post-stroke. Maximum microglial/macrophage activation occurred seven days post-stroke for both measures, and the diminished activation found after two weeks continued to four weeks. Our results suggest that nanoparticle-enhanced MRI may constitute a novel approach for monitoring the dynamic development of neuroinflammation in living animals during the progression and treatment of stroke.

Optogenetic Stimulation Reduces Neuronal Nitric Oxide Synthase Expression After Stroke

Abstract INTRODUCTION Poststroke optogenetic stimulation has been shown to enhance neurovascular coupling and functional recovery. Neuronal nitric oxide synthase (nNOS) has been implicated as a key regulator of neurovascular response in acute stroke but its role in subacute recovery remains unclear. Here we investigate nNOS expression in stroke mice undergoing optogenetic stimulation of the contralesional lateral cerebellar nucleus (cLCN). We also examine the effects of nNOS inhibition on functional recovery using a pharmacological inhibitor targeting nNOS. METHODS Transgenic Thy1-ChR2-YFP male mice (10-12 wk) were used. Stereotaxic surgery was performed to implant a fiber cannula in the cLCN and animals underwent intraluminal middle cerebral artery suture occlusion (30 min). Optogenetic stimulation began at poststroke (PD) day 5 and continued until PD14. Sensorimotor tests were used to assess behavioral recovery at PD4, 7, 10, and 14. At PD15, primary motor cortex from both ipsi- and contralesional motor cortex (iM1, cM1) were dissected. nNOS mRNA and protein levels were examined using quantitative polymerase chain reaction and western blot. In another set of studies, nNOS inhibitor ARL 17477 dihydrochloride (10 mg/kg, intraperitoneally) was administered daily between PD5-14 and functional recovery was evaluated using sensorimotor tests. RESULTS cLCN stimulated stroke mice demonstrated significant improvement in speed (cm/s) on the rotating beam task at PD10 and 14 day (P < .05, P < .001 respectively). nNOS mRNA and protein expression was significantly and selectively decreased in cM1 of cLCN stimulated mice (P < .05). The reduced nNOS expression in cM1 was negatively correlated with improved recovery (R2 = −0.839, Pearson P = .009). nNOS inhibitor-treated stroke mice exhibited a significant functional improvement in speed at PD10, when compared to stroke mice receiving vehicle (saline) (P < .05). CONCLUSION Our results suggest that nNOS may play a maladaptive role in poststroke recovery. Optogenetic stimulation of cLCN and systemic nNOS inhibition produce functional benefits after stroke.

Oophorectomy Reduces Estradiol Levels and Long-Term Spontaneous Neurovascular Recovery in a Female Rat Model of Focal Ischemic Stroke.

Although epidemiological evidence suggests significant sex and gender-based differences in stroke risk and recovery, females have been widely under-represented in preclinical stroke research. The neurovascular sequelae of brain ischemia in females, in particular, are largely uncertain. We set out to address this gap by a multimodal in vivo study of neurovascular recovery from endothelin-1 model of cortical focal-stroke in sham vs. ovariectomized female rats. Three weeks post ischemic insult, sham operated females recapitulated the phenotype previously reported in male rats in this model, of normalized resting perfusion but sustained peri-lesional cerebrovascular hyperreactivity. In contrast, ovariectomized (Ovx) females showed reduced peri-lesional resting blood flow, and elevated cerebrovascular responsivity to hypercapnia in the peri-lesional and contra-lateral cortices. Electrophysiological recordings showed an attenuation of theta to low-gamma phase-amplitude coupling in the peri-lesional tissue of Ovx animals, despite relative preservation of neuronal power. Further, this chronic stage neuronal network dysfunction was inversely correlated with serum estradiol concentration. Our pioneering data demonstrate dramatic differences in spontaneous recovery in the neurovascular unit between Ovx and Sham females in the chronic stage of stroke, underscoring the importance of considering hormonal-dependent aspects of the ischemic sequelae in the development of novel therapeutic approaches and patient recruitment in clinical trials.

Growth Differentiation Factor 11 Promotes Neurovascular Recovery After Stroke in Mice.

Background: Growth differentiation factor 11 (GDF11), a member of transforming growth factor-β (TGF-β) superfamily, was shown to rejuvenate cardiac and skeletal muscle function and to improve cerebral vasculature and neurogenesis in old mice. However, recent experimental data reported that raising GDF11 levels inhibited skeletal muscle regeneration and had no effect on cardiac hypertrophy. Our aim was to investigate the effects of GDF11 on brain repair during the recovery phase after stroke.Methods: Mice were subjected to distal middle cerebral artery occlusion, and recombinant GDF11 (rGDF11) was injected intraperitoneally once a day during days 7–13 after stroke. Neuronal precursor cells (NPCs) proliferation and angiogenesis were assayed at 14 days. Neuronal regeneration was assayed at 42 days. The beam-walking test and CatWalk were used to evaluate behavioral functions. Downstream pathways of GDF11 were also investigated.Results: GDF11 was upregulated in the ipsilateral peri-infarct cortex and subventricular zone (SVZ) at 14 days after stroke. Treatment with rGDF11 enhanced the number of newborn NPCs and endothelial cells, microvascular length and area, and brain capillary perfusion. Western blots showed that rGDF11 upregulated brain-derived neurotrophic factor (BDNF) and increased the levels of proangiogenic factor angiopoietin-2 (Ang-2) and phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2). We also found that rGDF11 upregulated the transcription factors Smad2 and Smad3 phosphorylation, but these activations were blocked by a TGF-β receptor inhibitor SB431542. Moreover, rGDF11-induced angiogenic remodeling and NPCs proliferation were reversed by injection of SB431542, suggesting that GDF11 may exert its effect via the TGF-β/Smad2/3 signaling pathway. Finally, treating mice with rGDF11 resulted in a significant increase in neuronal regeneration and functional recovery.Conclusion: GDF11 promoted neurogenesis and angiogenesis and contributed to functional recovery after stroke in mice.

Abstract WP421: Remote Ischemic Injury Mediates Hippocampal Astrocytic Endfeet Remodeling, Increases Vascular Injury and Worsens Cognitive Deficits in Diabetes

Diabetes increases the risk and severity of cognitive impairment, especially after ischemic stroke. We showed pathological rearrangement of cerebrovascular networks in the cortex and striatum is associated with poor neuronal restoration leading to worsened cognitive deficits in diabetes. However, little is known about the effect of diabetes on vascular architecture of hippocampus, a domain critical to memory and learning. We hypothesized that diabetes would mediate vasoneuronal remodeling and exacerbate post-stroke deficits in hippocampal tasks. Diabetes was induced by a high fat diet and low dose STZ in male Wistar rats. Cognition was assessed for 2 weeks after stroke (90-min middle cerebral artery occlusion MCAO) by Novel Object Recognition (NOR) and Placement (NPR), Y-Maze and Social Recognition tests. Vasoneuronal architecture and integrity were assessed by measurement of surface area, branch density, IgG extravasation, neuron counts and aquaporin4 (AQP4) polarity for astrocytic endfeet remodeling in dentate gyrus. Diabetes alone increased BBB permeability and unpolarized AQP4, which were exacerbated after MCAO. Post-stroke branch density was higher in control but lower in diabetic rats. While lower baseline hippocampal neuron counts in diabetes was not further reduced after MCAO, poor baseline cognitive and social performance deteriorated. These results suggest that remote ischemic injury can induce profound vasoglial remodeling in the hippocampus and worsens functional outcomes without reducing neurons. Understanding how diabetes impacts post-stroke neurovascular recovery in hippocampus may provide critical insight to worsened cognitive outcomes in this population.

Sirt3 Mediates the Inhibitory Effect of Adjudin on Astrocyte Activation and Glial Scar Formation following Ischemic Stroke.

In response to stroke-induced injury, astrocytes can be activated and form a scar. Inflammation is an essential component for glial scar formation. Previous study has shown that adjudin, a potential Sirt3 activator, could attenuate lipopolysaccharide (LPS)- and stroke-induced neuroinflammation. To investigate the potential inhibitory effect and mechanism of adjudin on astrocyte activation, we used a transient middle cerebral artery occlusion (tMCAO) model with or without adjudin treatment in wild type (WT) and Sirt3 knockout (KO) mice and performed a wound healing experiment in vitro. Both our in vivo and in vitro results showed that adjudin reduced astrocyte activation by upregulating Sirt3 expression. In addition, adjudin treatment after stroke promoted functional and neurovascular recovery accompanied with the decreased area of glial scar in WT mice, which was blunted by Sirt3 deficiency. Furthermore, adjudin could increase Foxo3a and inhibit Notch1 signaling pathway via Sirt3. Both the suppression of Foxo3a and overexpression of N1ICD could alleviate the inhibitory effect of adjudin in vitro indicating that Sirt3-Foxo3a and Sirt3-Notch1 signaling pathways were involved in the inhibitory effect of adjudin in wound healing experiment.

HMGB1 promotes neurovascular remodeling via Rage in the late phase of subarachnoid hemorrhage

High-mobility group box1 (HMGB1) is a nuclear protein widely expressed in the central nervous system. Extracellular HMGB1 serves as a proinflammatory cytokine and contributes to brain injury during the acute stage post-stroke. Recently, increasing evidence has demonstrated beneficial effects of HMGB1 in some types of brain injury, but little is known about its effects during the late phase of subarachnoid hemorrhage (SAH). This study was designed to explore the potential roles and mechanisms of HMGB1 and its receptor, receptor for advanced glycation end-products (Rage), on brain recovery in the late stage of experimental SAH. Two inhibitors of HMGB1, ethyl pyruvate and glycyrrhizin (EP and GA), and Rage antagonist FPS-ZM1 were used to determine whether HMGB1 promotes brain recovery after SAH. The administration of EP, GA, and FPS-ZM1 effectively reduced HMGB1 and Rage expression. Correspondingly, protein levels of beneficial growth factors (NGF, BDNF, and VEGF) and numbers of BrdU and DCX positive neurons in the cortex were also decreased. The biphasic roles of HMGB1 may be based on the different redox modifications of cysteine residues. In this research, rats injected with two different redox status HMGB1 showed different prognosises at 7–14day after SAH. Recombinant HMGB1 can promote cytokine stimulating activity and aggravate brain injury. However, oxidized HMGB1 was unable to stimulate TNF production but can promote brain recovery by promoting neurotrophin expression. In conclusion, our investigation identified that HMGB1 promotes neurovascular recovery via Rage and may act in the oxidized state in the late stage of SAH.

EphrinB2 Activation Enhances Vascular Repair Mechanisms and Reduces Brain Swelling After Mild Cerebral Ischemia

Cerebral edema caused by the disruption of the blood-brain barrier is a major complication after stroke. Therefore, strategies to accelerate and enhance neurovascular recovery after stroke are of prime interest. Our main aim was to study the role of ephrinB2/EphB4 signaling in mediating the vascular repair and in blood-brain barrier restoration after mild cerebral ischemia occlusion/reperfusion. Here, we show that the guidance molecule ephrinB2 plays a key role in neurovascular protection and blood-brain barrier restoration after stroke. In a focal stroke model, we characterize the stroke-induced damage to cerebral blood vessels and their subsequent endogenous repair on a cellular, molecular, and functional level. EphrinB2 and its tyrosine kinase receptor EphB4 are upregulated early after stroke by endothelial cells and perivascular support cells, in parallel to their reassembly during neurovascular recovery. Using both retroviral and pharmacological approaches, we show that the inhibition of ephrinB2/EphB4 signaling suppresses post-middle cerebral artery occlusion neurovascular repair mechanisms resulting in an aggravation of brain swelling. In contrast, the activation of ephrinB2 after brain ischemia leads to an increased pericyte recruitment and increased endothelial-pericyte interaction, resulting in an accelerated neurovascular repair after ischemia. We show that reducing swelling could result in improved outcome because of reduction in damaged brain tissue. We also identify a novel role for ephrinB2/EphB4 signaling in the maintenance of the neurovascular homeostasis and provide a novel therapeutic approach in reducing brain swelling after stroke.

Promoting Neurovascular Recovery in Aged Mice after Ischemic Stroke - Prophylactic Effect of Omega-3 Polyunsaturated Fatty Acids.

The aged population is among the highest at risk for ischemic stroke, yet most stroke patients of advanced ages (>80 years) are excluded from access to thrombolytic treatment by tissue plasminogen activator, the only FDA approved pharmacological therapy for stroke victims. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) robustly alleviate ischemic brain injury in young adult rodents, but have not yet been studied in aged animals. This study investigated whether chronic dietary supplementation of n-3 PUFAs protects aging brain against cerebral ischemia and improves long-term neurological outcomes. Aged (18-month-old) mice were administered n-3 PUFA-enriched fish oil in daily chow for 3 months before and up to 8 weeks after 45 minutes of transient middle cerebral artery occlusion (tMCAO). Sensorimotor outcomes were assessed by cylinder test and corner test up to 35 days and brain repair dynamics evaluated immunohistologically up to 56 days after tMCAO. Mice receiving dietary supplementation of n-3 PUFAs for 3 months showed significant increases in brain ratio of n-3/n-6 PUFA contents, and markedly reduced long-term sensorimotor deficits and chronic ischemic brain tissue loss after tMCAO. Mechanistically, n-3 PUFAs robustly promoted post-ischemic angiogenesis and neurogenesis, and enhanced white matter integrity after tMCAO. The Pearson linear regression analysis revealed that the enhancement of neurogenesis and white matter integrity both correlated positively with improved sensorimotor activities after tMCAO. This study demonstrates that prophylactic dietary supplementation of n-3 PUFAs effectively improves long-term stroke outcomes in aged mice, perhaps by promoting post-stroke brain repair processes such as angiogenesis, neurogenesis, and white matter restoration.

Chronic monitoring of vascular progression after ischemic stroke using multiexposure speckle imaging and two-photon fluorescence microscopy.

Monitoring the progression of the vascular structure and cerebral blood flow (CBF) after brain injury is vital to understand the neurovascular recovery process. Multiexposure speckle imaging (MESI) provides a quantitatively accurate technique for chronically measuring the postocclusion CBF perfusion of the infarct and peri-infarct regions in rodent stroke models, while multiphoton microscopy offers direct visualization of the microvascular structure. In this paper, we present imaging outcomes extending 35 days after photo-thrombotic occlusion, tracking the progression of the vasculature throughout this period. We compare MESI flow estimates within the unresolvable parenchyma with subsurface microvascular volume fractions taken with two-photon microscopy in the same regions to assess how the vascular density influences the surface-integrated MESI flow values. The MESI flow measurements and volume fractions are shown to have high correlations (r=0.90) within areas of recovering vasculature in the peri-infarct region. We also observe vascular reorientation occurring within the microvascular structure throughout the 35-day postocclusion period. With the combination of a chronic mouse model and relatively noninvasive optical imaging techniques, we present an imaging protocol for monitoring long-term vascular progression after photo-thrombotic occlusion with the potential to test the efficacy of rehabilitation and pharmacological therapies.

Lipocalin‐2 enhances angiogenesis in rat brain endothelial cells via reactive oxygen species and iron‐dependent mechanisms

Inflammation is a key part of central nervous system pathophysiology. However, inflammatory factors are now thought to have both beneficial and deleterious effects. Here, we examine the hypothesis that lipocalin-2 (LCN2), an inflammatory molecule that can be up-regulated in the distressed central nervous system, may enhance angiogenesis in brain endothelial cells. Adding LCN2 (0.5-2.0 μg/mL) to RBE (Rat brain endothelial cells). 4 rat brain endothelial cells significantly increased matrigel tube formation and scratch migration, and also elevated levels of iron and reactive oxygen species. Co-treatment with a radical scavenger (U83836E), a Nox inhibitor (apocynin) and an iron chelating agent (deferiprone) significantly dampened the ability of LCN2 to enhance tube formation and scratch migration in brain endothelial cells. These findings provide in vitro proof of the concept that LCN2 can promote angiogenesis via iron- and reactive oxygen species-related pathways, and support the idea that LCN2 may contribute to the neurovascular recovery aspects of inflammation. Angiogenesis is an important part of stroke recovery. In the present study, we examined the hypothesis that lipocalin-2 (LCN2) may enhance angiogenesis in brain endothelial cells. LCN2 promoted tube formation and migration via iron and ROS-related pathways in rat brain endothelial cells. ROS scavengers, Nox inhibitors and iron chelators all dampened the ability of LCN2 to enhance in vitro angiogenesis. These findings support the idea that LCN2 that is released by damaged neurons may act as a 'help me' signal that promotes neurovascular recovery after stroke and brain injury.

Neurovascular Recovery via Cotransplanted Neural and Vascular Progenitors Leads to Improved Functional Restoration after Ischemic Stroke in Rats

SummaryThe concept of the “neurovascular unit,” emphasizing the interactions between neural and vascular components in the brain, raised the notion that neural progenitor cell (NPC) transplantation therapy aimed at neural repair may be insufficient for the treatment of ischemic stroke. Here, we demonstrate that enhanced neurovascular recovery via cotransplantation of NPCs and embryonic stem cell-derived vascular progenitor cells (VPCs) in a rat stroke model is correlated with improved functional recovery after stroke. We found that cotransplantation promoted the survival, migration, differentiation, and maturation of neuronal and vascular cells derived from the cotransplanted progenitors. Furthermore, it triggered an increased generation of VEGF-, BDNF-, and IGF1-expressing neural cells derived from the grafted NPCs. Consistently, compared with transplantation of NPCs alone, cotransplantation more effectively improved the neurobehavioral deficits and attenuated the infarct volume. Thus, cotransplantation of NPCs and VPCs represents a more effective therapeutic strategy for the treatment of stroke than transplantation of NPCs alone.

Postacute stromal cell-derived factor-1α expression promotes neurovascular recovery in ischemic mice.

Acute interventions of stroke are often challenged by a narrow treatment window. In this study, we explore treatments in the postacute phase of stroke with wider windows of opportunity. We investigated the effects of stromal cell-derived factor (SDF-1α) in neurovascular recovery during the postacute phase and downstream signaling pathways, underlying SDF-1α-mediated neurovascular recovery. Adult male Institute of Cancer Research (ICR) mice underwent middle cerebral artery occlusion. One week after middle cerebral artery occlusion, the animals received stereotactic injection of adenoassociated virus (AAV) carrying SDF-1α gene as treatment or AAV-green fluorescent protein as control and were monitored for 5 weeks. Neurobehavioral outcomes were evaluated, and brain atrophy was measured. Neurogenesis and angiogenesis were examined. The proliferation and migration of neural progenitor cells were evaluated. Downstream pathways of SDF-1α were investigated. Inflammatory response was monitored. Neurobehavioral outcomes were improved, and brain atrophy was greatly reduced for ≤5 weeks in AAV-SDF-1α groups when compared with the control. SDF-1 receptor CXCR4 was upregulated and colocalized with neural and endothelial progenitor cells. The number of nestin(+) and doublecortin(+)/bromodeoxyuridine(+) cells in the subventricular zone, doublecortin(+) and neuron(+)/bromodeoxyuridine(+) cells in the perifocal region, and cluster of differentiation (CD)31(+) and bromodeoxyuridine(+)/CD31(+) microvessels are also significantly increased in AAV-SDF-1α groups. Administration of CXCR4 antagonist AMD3100 eliminated the beneficial effects of SDF-1α. SDF-1α/CXCR4 interaction activated AKT, extracellular signal-regulated kinases (ERK), and P38 mitogen-activated protein kinase (MAPK) signaling pathways but not the c-Jun N-terminal kinase (JNK) pathway. SDF-1α promoted neurogenesis and angiogenesis during the postacute phase of ischemia without eliciting an inflammatory response. AAV-SDF-1α expression represents a promising avenue for ischemic stroke therapy with a wider treatment window.

Reactive astrocytes promote adhesive interactions between brain endothelium and endothelial progenitor cells via HMGB1 and beta-2 integrin signaling

Endothelial progenitor cells (EPCs) may contribute to neurovascular repair after stroke and neurodegeneration. A key step in this process should involve adhesive interactions between EPCs and the targeted cerebral endothelium. Here, we tested the hypothesis that reactive astrocytes may play a critical role in enhancing adhesive interactions and transmigration of EPCs across cerebral endothelial cells. Transiently seeding EPCs onto a monolayer of RBE.4 rat brain endothelial cells resulted in a time-dependent adherence between the two cell types. Blocking β2 integrins on EPCs or blocking the receptor for advanced glycation endproducts (RAGE) on endothelial cells significantly decreased EPC-endothelial adherence. Next, we tested whether reactive astrocytes can enhance this process by growing EPCs, brain endothelial cells and astrocytes together in a transwell co-culture system. The presence of reactive astrocytes in the lower chamber significantly promoted adherence between EPCs and endothelial cells in the upper chamber. This process involved the release of soluble HMGB1 from reactive astrocytes that then upregulated endothelial expression of RAGE via Egr1 signaling. Directly adding HMGB1 to the transwell system also promoted EPC-endothelial adhesion and accelerated EPC transmigration into the lower chamber. These initial findings provide proof-of-concept that reactive astrocytes promote crosstalk between cerebral endothelium and EPCs. Further investigation of this phenomenon may lead to a better understanding of cell–cell interactions required for neurovascular recovery after stroke.

Effects of normobaric oxygen on the progression of focal cerebral ischemia in rats

Normobaric oxygen (NBO) reduces infarction at 24–48h in experimental models of focal cerebral ischemia. However, to be clinically relevant, longer term safety and efficacy must be explored. Here, we assessed the effects of NBO on glial activation, neurovascular recovery, and behavioral outcomes at 2weeks after transient focal ischemia in rats. 100min transient focal ischemia was induced by intraluminal occlusion of the middle cerebral artery in adult male Sprague–Dawley rats. Animals were randomized into sham, controls or 85′NBO started 15min after ischemic onset. Infarct volumes and behavioral outcomes were blindly quantified. Immunohistochemistry was used to examine the effects of NBO on glial activation and neurovascular responses. After 2weeks of reperfusion the infarct volume was marked reduced in animals subjected to NBO. They also had better outcomes in forelimb placement test and in body-swing test and weight loss reduction. After 14days, NBO decreased expression of Iba1, a marker of activated microglia, and GFAP, a marker of activated astrocytes. NBO treatment had no detectable effect on angiogenesis. These results suggest that protective effects of NBO may persist for up to 2weeks post-stroke.

Potential Roles of HDAC Inhibitors in Mitigating Ischemia-induced Brain Damage and Facilitating Endogenous Regeneration and Recovery

Ischemic stroke is a leading cause of death and disability worldwide, with few available treatment options. The pathophysiology of cerebral ischemia involves both early phase tissue damage, characterized by neuronal death, inflammation, and blood-brain barrier breakdown, followed by late phase neurovascular recovery. It is becoming clear that any promising treatment strategy must target multiple points in the evolution of ischemic injury to provide substantial therapeutic benefit. Histone deacetylase (HDAC) inhibitors are a class of drugs that increase the acetylation of histone and non-histone proteins to activate transcription, enhance gene expression, and modify the function of target proteins. Acetylation homeostasis is often disrupted in neurological conditions, and accumulating evidence suggests that HDAC inhibitors have robust protective properties in many preclinical models of these disorders, including ischemic stroke. Specifically, HDAC inhibitors such as trichostatin A, valproic acid, sodium butyrate, sodium 4-phenylbutyrate, and suberoylanilide hydroxamic acid have been shown to provide robust protection against excitotoxicity, oxidative stress, ER stress, apoptosis, inflammation, and bloodbrain barrier breakdown. Concurrently, these agents can also promote angiogenesis, neurogenesis and stem cell migration to dramatically reduce infarct volume and improve functional recovery after experimental cerebral ischemia. In the following review, we discuss the mechanisms by which HDAC inhibitors exert these protective effects and provide evidence for their strong potential to ultimately improve stroke outcome in patients.

Abstract TP117: MiR-15a/16-1 Cluster Inhibits Angiogenesis In Mouse After Ischemic Stroke

Angiogenesis may occur as a natural defense response against neurological diseases. Accumulating studies have shown that post-ischemic angiogenesis promotes functional recovery after ischemic stroke and is related to longer survival time in ischemic stroke patients. MicroRNAs (miRs) have been documented as a novel family of noncoding small RNAs that negatively modulate protein expression in various organisms. Recent studies have revealed important roles for miRs in regulating angiogenesis. However, the function of miRs in the angiogenic processes after ischemic stroke is unknown. We have previously demonstrated that the miR-15a/16-1 cluster has an anti-survival role in ischemia-induced endothelial cell death. Here we further investigate the role of the miR-15a/16-1 cluster in the regulation of post-ischemic angiogenesis. We have shown that expression of the miR-15a/16-1 cluster is significantly increased in the cerebral vasculature at the penumbral area 7 days after mouse middle cerebral artery occlusion (MCAO). Accordingly, in comparison with the wild-type animals, endothelial cell (EC)-selective miR-15a/16-1 transgenic overexpression leads to reduced cerebral blood vessel formation, increased brain infarction and neurological deficits in mice 7 days post-MCAO. Mechanistically, lentivirus-mediated miR-15a/16-1 gain- or loss-of-function reduces or increases FGFR1 and VEGF mRNA or protein levels in mouse cerebral vascular endothelial cell cultures, respectively. Through the bioinformatics analysis, 3’UTR luciferase reporter assays, and immunoassays, we further found that miR-15a and miR-16-1 bind to the 3’UTR of FGFR1 or VEGF mRNA, and directly inhibit FGFR1 and VEGF activities. Our findings suggest the miR-15a/16-1 cluster can suppress post-ischemic cerebral angiogenesis through direct inhibition of endothelial FGFR1 and VEGF activities. Elucidating the molecular mechanism of miR-15a/16-1 cluster-mediated angiogenesis will provide new insights into the understanding of miR function in the post-ischemic neurovascular remodeling and neurological recovery and open a new area with potential promise for further development of neurorestorative therapies after ischemic stroke.

TMP Prevents Retinal Neovascularization and Imparts Neuroprotection in an Oxygen-Induced Retinopathy Model

To evaluate the effects of tetramethylpyrazine (TMP) on retinal neovascularization (NV) and neuroprotection in an oxygen-induced retinopathy (OIR) model. Neonatal C57BL/6J mice were subjected to 75% oxygen from postnatal day 7 (P7) to P12 and then returned to room air. TMP (200 mg/kg) or normal saline was given daily from P12 to P17. Immunostaining, HE staining, TUNEL assay, and RT-PCR were used to assess the effects of TMP on retinal neurovascular repair. TMP effectively prevented pathologic NV and accelerated physiologic revascularization by enhancing the formation of endothelial tip cells at the edges of the repairing capillary networks and preserving the astrocytic template in the avascular retina. TMP also prevented morphologic changes and significantly decreased TUNEL-positive cells in the avascular retina by rescuing neurons such as amacrine, rod bipolar, horizontal, and Müller cells. In TMP-treated mice retinas, there was a less obvious loss of amacrine cell bodies and their distinct bands; the number of both rod bipolar and horizontal cell bodies, as well as the density of their dendrites in the outer plexiform layer, was greater than that in OIR control mice. TMP not only decreased the loss of alignment of Müller cell bodies and distortion of processes but reduced the reactive expression of GFAP in Müller cells. Furthermore, HIF-1α and VEGF mRNA expression were downregulated in TMP-treated mice retinas. TMP improved neurovascular recovery by preventing NV and protecting retinal astroglia cells and neurons from ischemia-induced cell death partially due to its downregulation of HIF-1α and VEGF mRNA expression.

Pro-Angiogenic Effects of Resveratrol in Brain Endothelial Cells: Nitric Oxide-Mediated Regulation of Vascular Endothelial Growth Factor and Metalloproteinases

Resveratrol may be a powerful way of protecting the brain against a wide variety of stress and injury. Recently, it has been proposed that resveratrol not only reduces brain injury but also promotes recovery after stroke. But the underlying mechanisms are unclear. Here, we tested the hypothesis that resveratrol promotes angiogenesis in cerebral endothelial cells and dissected the signaling pathways involved. Treatment of cerebral endothelial cells with resveratrol promoted proliferation, migration, and tube formation in Matrigel assays. Consistent with these pro-angiogenic responses, resveratrol altered endothelial morphology resulting in cytoskeletal rearrangements of β-catenin and VE-cadherin. These effects of resveratrol were accompanied by activation of phosphoinositide 3 kinase (PI3-K)/Akt and Mitogen-Activated Protein Kinase (MAPK)/ERK signaling pathways that led to endothelial nitric oxide synthase upregulation and increased nitric oxide (NO) levels. Subsequently, elevated NO signaling increased vascular endothelial growth factor and matrix metalloproteinase levels. Sequential blockade of these signaling steps prevented resveratrol-induced angiogenesis in cerebral endothelial cells. These findings provide a mechanistic basis for the potential use of resveratrol as a candidate therapy to promote angiogenesis and neurovascular recovery after stroke.