Peri-ischemic Area Research Articles

Neurovascular Uncoupling Is Linked to Microcirculatory Dysfunction in Regions Outside the Ischemic Core Following Ischemic Stroke.

Background Normal brain function depends on the ability of the vasculature to increase blood flow to regions with high metabolic demands. Impaired neurovascular coupling, such as the local hyperemic response to neuronal activity, may contribute to poor neurological outcome after stroke despite successful recanalization, that is, futile recanalization. Methods and Results Mice implanted with chronic cranial windows were trained for awake head-fixation before experiments. One-hour occlusion of the anterior middle cerebral artery branch was induced using single-vessel photothrombosis. Cerebral perfusion and neurovascular coupling were assessed by optical coherence tomography and laser speckle contrast imaging. Capillaries and pericytes were studied in perfusion-fixed tissue by labeling lectin and platelet-derived growth factor receptor β. Arterial occlusion induced multiple spreading depolarizations over 1 hour associated with substantially reduced blood flow in the peri-ischemic cortex. Approximately half of the capillaries in the peri-ischemic area were no longer perfused at the 3- and 24-hour follow-up (45% [95% CI, 33%-58%] and 53% [95% CI, 39%-66%] reduction, respectively; P<0.0001), which was associated with contraction of an equivalent proportion of peri-ischemic capillary pericytes. The capillaries in the peri-ischemic cortex that remained perfused showed increased point prevalence of dynamic flow stalling (0.5% [95% CI, 0.2%-0.7%] at baseline, 5.1% [95% CI, 3.2%-6.5%] and 3.2% [95% CI, 1.1%-5.3%] at 3- and 24-hour follow-up, respectively; P=0.001). Whisker stimulation at the 3- and 24-hour follow-up led to reduced neurovascular coupling responses in the sensory cortex corresponding to the peri-ischemic region compared with that observed at baseline. Conclusions Arterial occlusion led to contraction of capillary pericytes and capillary flow stalling in the peri-ischemic cortex. Capillary dysfunction was associated with neurovascular uncoupling. Neurovascular coupling impairment associated with capillary dysfunction may be a mechanism that contributes to futile recanalization. Hence, the results from this study suggest a novel treatment target to improve neurological outcome after stroke.

Longitudinal Synaptic Density PET with 11 C-UCB-J 6Months After Ischemic Stroke.

The purpose of this study was to explore longitudinal changes in synaptic density after ischemic stroke in vivo with synaptic vesicle protein 2A (SV2A) positron emission tomography (PET). We recruited patients with an ischemic stroke to undergo 11 C-UCB-J PET/MR within the first month and 6 months after the stroke. We investigated longitudinal changes of partial volume corrected 11 C-UCB-J standardized uptake value ratio (SUVR; relative to centrum semiovale) within the ischemic lesion, peri-ischemic area and unaffected ipsilesional and contralesional grey matter. We also explored crossed cerebellar diaschisis at 6 months. Additionally, we defined brain regions potentially influencing upper limb motor recovery after stroke and studied 11 C-UCB-J SUVR evolution in comparison to baseline. In 13 patients (age=67 ± 15 years) we observed decreasing 11 C-UCB-J SUVR in the ischemic lesion (ΔSUVR=-1.0, p= 0.001) and peri-ischemic area (ΔSUVR=-0.31, p= 0.02) at 6 months after stroke compared to baseline. Crossed cerebellar diaschisis as measured with 11 C-UCB-J SUVR was present in 11 of 13 (85%) patients at 6 months. The 11 C-UCB-J SUVR did not augment in ipsilesional or contralesional brain regions associated with motor recovery. On the contrary, there was an overall trend of declining 11 C-UCB-J SUVR in these brain regions, reaching statistical significance only in the nonlesioned part of the ipsilesional supplementary motor area (ΔSUVR=-0.83, p= 0.046). At 6 months after stroke, synaptic density further declined in the ischemic lesion and peri-ischemic area compared to baseline. Brain regions previously demonstrated to be associated with motor recovery after stroke did not show increases in synaptic density. ANN NEUROL 2023;93:911-921.

In Vivo Detection of Neurofibrillary Tangles by 18F-MK-6240 PET/MR in Patients With Ischemic Stroke.

The risk of developing Alzheimer disease is increased after stroke, and this association may not solely be driven by traditional vascular risk factors. Neuronal death leads to the release of tau proteins, which can become dephosphorylated, rephosphorylated, or hyperphosphorylated in the setting of ischemia, possibly leading to formation of neurofibrillary tangles (NFT). Therefore, a potential synergistic effect between development of tauopathy and cerebrovascular lesion burden may contribute to cognitive decline after stroke. We explored the spatial and temporal distribution of NFT after ischemic stroke in vivo by using 18F-MK-6240 PET. We included patients with a first ischemic stroke to undergo longitudinal 18F-MK-6240 PET/MR within 2-4 weeks and 6 months after stroke. For cross-sectional analyses, we also included age-matched healthy controls. We delineated 5 volumes of interest based on T2 FLAIR and T1 MR data: the ischemic lesion, 3 consecutive peri-ischemic areas, and the remaining ipsilesional hemisphere. We performed region-based voxel-wise partial volume correction on the PET data and calculated standardized uptake value ratios (SUVRs) with the cerebellum as the reference region. We did not quantify PET scans of patients within the first month after stroke (n = 17; median age 73 years [interquartile range {IQR}: 62-82 years]) because the signal intensity was influenced by blood-brain barrier breakdown hampering a reliable data analysis. At 6 months after the event (n = 13; median age 71 years [IQR: 60-79 years]), 18F-MK-6240 SUVR was increased in the ischemic lesion compared with 20 age-matched healthy controls (median age 71.5 years [IQR: 66-76 years]; ratiolesion/controls = 1.62 ± 0.54; 1-sample t test: p = 0.0015) and gradually decreased in the surrounding tissue (1-way within-subject analysis of variance [F{1.2, 14.8} = 18.0, p = 0.00043]). These findings suggest that NFT may form after ischemic stroke and spread in the peri-ischemic brain parenchyma. Further follow-up is required to gain more insight into the spatial and temporal dynamics of this tauopathy after ischemic stroke.

Establishment of the reproducible branch retinal artery occlusion mouse model and intravital longitudinal imaging of the retinal CX3CR1-GFP+ cells after spontaneous arterial recanalization.

Animal models of retinal artery occlusion (RAO) have been widely used in many studies. However, most of these studies prefer using a central retinal artery occlusion (CRAO) which is a typical global ischemia model of the retina, due to the technical limitation of producing single vessel targeted modeling with real-time imaging. A focal ischemia model, such as branch retinal artery occlusion (BRAO), is also needed for explaining interactions, including the immunological reaction between the ischemic retina and adjacent healthy retina. Accordingly, a relevant model for clinical RAO patients has been demanded to understand the pathophysiology of the RAO disease. Herein, we establish a convenient BRAO mouse model to research the focal reaction of the retina. As a photo-thrombotic agent, Rose bengal was intravenously injected into 7 week-old transgenic mice (CX3CR1-GFP) for making embolism occlusion, which causes pathology similarly to clinical cases. In an optimized condition, a 561 nm laser (13.1 mw) was projected to a targeted vessel to induce photo-thrombosis for 27 s by custom-built retinal confocal microscopy. Compared to previous BRAO models, the procedures of thrombosis generation were naturally and minimal invasively generated with real-time retinal imaging. In addition, by utilizing the self-remission characteristics of Rose bengal thrombus, a reflow of the BRAO with immunological reactions of the CX3CR1-GFP+ inflammatory cells such as the retinal microglia and monocytes was monitored and analyzed. In this models, reperfusion began on day 3 after modeling. Simultaneously, the activation of CX3CR1-GFP+ inflammatory cells, including the increase of activation marker and morphologic change, was confirmed by immunohistochemical (IHC) staining and quantitative real-time PCR. CD86 and Nox2 were prominently expressed on day 3 after the modeling. At day 7, blood flow was almost restored in the large vessels. CX3CR1-GFP+ populations in both superficial and deep layers of the retina also increased around even in the BRAO peri-ischemic area. In summary, this study successfully establishes a reproducible BRAO modeling method with convenient capabilities of easily controllable time points and selection of a specific single vessel. It can be a useful tool to analyze the behavior of inflammatory cell after spontaneous arterial recanalization in BRAO and further investigate the pathophysiology of BRAO.

Up-regulation of sphingosine-1-phosphate receptors and sphingosine kinase 1 in the peri-ischemic area after transient middle cerebral artery occlusion in mice

There is thought to be a strong relationship between sphingosine-1-phosphate (S1P) signaling and pathophysiolosy of cerebral ischemia. We examined the change of expression and distribution of S1P receptors (S1PRs) and sphingosine kinases (SphKs) after cerebral ischemia in male C57BL6/J mice using immunohistochemical analysis at 1, 5, 14, and 28 days after 30 min of transient middle cerebral artery occlusion (tMCAO). S1PR1, 3, and 5 were transiently induced in the cells, which were morphologically similar to neurons in the peri-infarct lesion with a peak seen at 1 day after tMCAO (p < 0.01 vs. sham control). S1PR2 appeared in the inner layer of vessels in the ischemic core (p < 0.01 vs. sham control) and the peri-infarct lesion (p < 0.01 vs. sham control) at the acute phase after tMCAO. However, SphK1 was strongly induced at 1 and 5 days after tMCAO (p < 0.01 vs. sham control) in the peri-infarct lesion, whereas SphK2 expression did not change. Western blot analysis at 1 and 5 days after 30 min of tMCAO revealed that the expression of S1PRs were transiently enhanced at the acute phase, which was consistent with the immunohistochemical results. Double immunofluorescent analysis revealed S1PR2/NG2- and S1PR2/CD31-, S1PR3/CD31-, and S1PR5/CD31-double positive cells in the peri-infarct lesion 1 day after tMCAO. The present results suggest that S1PRs and SphK1 may be important therapeutic targets for rescuing the peri-infarct lesion.

A temporal change of in vivo oxidative stress imaging in a mouse stroke model

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a pivotal role in cellular defense against oxidative stress damage after ischemic stroke. In the present study, we examined the time-dependent change of in vivo optical imaging of oxidative stress after stroke with Keap1 dependent oxidative stress detector (OKD) mice. OKD mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 45min, and in vivo optical signals were detected during the pre-operative period, 12 h, 1 d, 3 d, and 7 d after tMCAO. Ex vivo imaging was performed immediately after obtaining in vivo optical signals at 1 d after tMCAO. Immunohistochemical analyses and infarct volume were also examined after in vivo imaging at each period. The in vivo signals showed a peak at 1 d after tMCAO that was slightly correlated to infarct volume. The strong ex vivo signals, which were detected in the peri-ischemic area, corresponded to endogenous Nrf2 expression. Moreover, endogenous Nrf2 expression was detected mainly in neurons followed by oligodendrocytes and pericytes, but only slightly in astrocytes, microglia, endothelial cells. The present study successfully demonstrated the temporal change of in vivo imaging of oxidative stress after tMCAO, which is consistent with strong expression of endogenous Nrf2 in the peri-ischemic area with a similar time course.

Peripheral Administration of IL-13 Induces Anti-inflammatory Microglial/Macrophage Responses and Provides Neuroprotection in Ischemic Stroke

Neuroinflammation is strongly induced by cerebral ischemia. The early phase after the onset of ischemic stroke is characterized by acute neuronal injury, microglial activation, and subsequent infiltration of blood-derived inflammatory cells, including macrophages. Therefore, modulation of the microglial/macrophage responses has increasingly gained interest as a potential therapeutic approach for the ischemic stroke. In our study, we investigated the effects of peripherally administered interleukin 13 (IL-13) in a mouse model of permanent middle cerebral artery occlusion (pMCAo). Systemic administration of IL-13 immediately after the ischemic insult significantly reduced the lesion volume, alleviated the infiltration of CD45+ leukocytes, and promoted the microglia/macrophage alternative activation within the ischemic region, as determined by arginase 1 (Arg1) immunoreactivity at 3 days post-ischemia (dpi). Moreover, IL-13 enhanced the expression of M2a alternative activation markers Arg1 and Ym1 in the peri-ischemic (PI) area, as well as increased plasma IL-6 and IL-10 levels at 3 dpi. Furthermore, IL-13 treatment ameliorated gait disturbances at day 7 and 14 and sensorimotor deficits at day 14 post-ischemia, as analyzed by the CatWalk gait analysis system and adhesive removal test, respectively. Finally, IL-13 treatment decreased neuronal cell death in a coculture model of neuroinflammation with RAW 264.7 macrophages. Taken together, delivery of IL-13 enhances microglial/macrophage anti-inflammatory responses in vivo and in vitro, decreases ischemia-induced brain cell death, and improves sensory and motor functions in the pMCAo mouse model of cerebral ischemia.

Visualization of ischemic stroke-related changes on 18F-THK-5351 positron emission tomography

BackgroundThe 18F-THK-5351 radiotracer has been used to detect the in vivo tau protein distribution in patients with tauopathy, such as Alzheimer’s disease and corticobasal syndrome. In addition, 18F-THK-5351 can also monitor neuroinflammatory process due to high affinity to astrogliosis. We aimed to explore 18F-THK-5351 distribution patterns and characteristics in patients with recent ischemic stroke.ResultsFifteen patients received 18F-THK-5351 positron emission tomography (PET) and diffusion tensor imaging (DTI) approximately 3 months after ischemic stroke. A region of interest (ROI) was placed in the peri-ischemic area and was mirrored on the contralateral side as the control, and a proportional value was derived from the ratio of the peri-ischemic ROI value over the mirrored ROI value. Increased 18F-THK-5351 retention was observed in the areas around and remote from the stroke location. The proportional 18F-THK-5351 values were negatively correlated with the proportional fractional anisotropy values (r = − 0.39, P = 0.04).Conclusion18F-THK-5351 PET imaging provides a potential tool for in vivo visualization of the widespread ischemia-related changes associated with a microstructural disruption in recent ischemic stroke patients.

Sulfosuccinimidyl oleate sodium is neuroprotective and alleviates stroke-induced neuroinflammation

BackgroundIschemic stroke is one of the main causes of death and disability worldwide. It is caused by the cessation of cerebral blood flow resulting in the insufficient delivery of glucose and oxygen to the neural tissue. The inflammatory response initiated by ischemic stroke in order to restore tissue homeostasis in the acute phase of stroke contributes to delayed brain damage.MethodsBy using in vitro models of neuroinflammation and in vivo model of permanent middle cerebral artery occlusion, we demonstrate the neuroprotective and anti-inflammatory effects of sulfosuccinimidyl oleate sodium (SSO).ResultsSSO significantly reduced the lipopolysaccharide/interferon-γ-induced production of nitric oxide, interleukin-6 and tumor necrosis factor-α, and the protein levels of inflammatory enzymes including nitric oxide synthase 2, cyclooxygenase-2 (COX-2), and p38 mitogen-activated protein kinase (MAPK) in microglia, without causing cell toxicity. Although SSO failed to directly alleviate glutamate-induced excitotoxicity in murine cortical neurons, it prevented inflammation-induced neuronal death in microglia-neuron co-cultures. Importantly, oral administration of SSO in Balb/c mice subjected to permanent occlusion of the middle cerebral artery reduced microglial activation in the peri-ischemic area and attenuated brain damage. This in vivo neuroprotective effect of SSO was associated with a reduction in the COX-2 and heme oxygenase-1 immunoreactivities.ConclusionsOur results suggest that SSO is an anti-inflammatory and a possible therapeutic candidate in diseases such as stroke where inflammation is a central hallmark.

Electroacupuncture Suppresses the NF-κB Signaling Pathway by Upregulating Cylindromatosis to Alleviate Inflammatory Injury in Cerebral Ischemia/Reperfusion Rats.

Electroacupuncture (EA) may reduce inflammatory injury by inhibiting nuclear factor-kappa B (NF-κB) signaling pathway activation after ischemic stroke. Thus, we explored temporal and spatial expression of cylindromatosis (CYLD), a negative feedback inhibitor of the NF-κB signaling pathway, to learn whether CYLD is essential for EA and reduction of inflammatory injury after focal cerebral ischemia/reperfusion. A middle cerebral artery occlusion/reperfusion (MCAO/R) model was established in male Sprague-Dawley (SD) rats and CYLD gene interference was used to investigate a potential role of neuroprotection. Rats were treated with EA (1 mA, 20 Hz for 5 min, 2 Hz for 30 min) at Baihui (GV 20), Hegu (LI 4) and Taichong (LR 3) acupoints, once daily, beginning 2 h after focal cerebral ischemia. Microglial activation and co-expression of CYLD and NF-κB were measured with immunofluorescence. Neuronal CX3CL1 expression was assayed to investigate the role of EA in the interaction between neurons and microglia via upregulation of CYLD. Then, CYLD, NF-κB p65 and p-IκBα protein expression was measured with Western blot. CYLD was mainly expressed in neurons of the peri-ischemic area after MCAO/R in rats and EA upregulated CYLD mRNA and protein from 24 to 72 h after focal cerebral ischemia/reperfusion. In addition, CYLD overexpression was positively correlated to neurobehavior and negatively connected with infarct volume and pro-inflammatory cytokines (TNF-α and IL-1β). Upregulation of CYLD by EA prevented NF-κB nuclear translocation and inhibition of neuronal CX3CL1 expression, which repressed activation of microglia. Finally, CYLD silencing significantly weakened suppression of the NF-κB signaling pathway by EA. In conclusion, upregulation of CYLD may underlie how EA could alleviate inflammatory injury after focal cerebral ischemia/reperfusion.

Time-dependent change of in vivo optical imaging of oxidative stress in a mouse stroke model.

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a pivotal role in cellular defense against oxidative stress damage after ischemic stroke. In the present study, we examined the time-dependent change of in vivo optical imaging of oxidative stress after stroke with Keap1-dependent oxidative stress detector (OKD) mice. OKD mice were subjected to transient middle cerebral artery occlusion (tMCAO) for 45 min, and in vivo optical signals were detected during the pre-operative period, 12 h, 1 d, 3 d, and 7 d after tMCAO. Ex vivo imaging was performed immediately after obtaining in vivo optical signals at 1 d after tMCAO. Immunohistochemical analyses and infarct volume were also examined after in vivo imaging at each period. The in vivo signals showed a peak at 1 d after tMCAO that was slightly correlated to infarct volume. The strong ex vivo signals, which were detected in the peri-ischemic area, corresponded to endogenous Nrf2 expression. Moreover, endogenous Nrf2 expression was detected mainly in neurons followed by oligodendrocytes and pericytes, but only slightly in astrocytes, microglia, endothelial cells. The present study successfully demonstrated the temporal change of in vivo imaging of oxidative stress after tMCAO, which is consistent with strong expression of endogenous Nrf2 in the peri-ischemic area with a similar time course. © 2017 Wiley Periodicals, Inc.

Dynamic mislocalizations of nuclear pore complex proteins after focal cerebral ischemia in rat.

Nuclear pore complexes (NPCs) play an important role in coordinating the transport of proteins and nucleic acids between the nucleus and cytoplasm, and are therefore essential for maintaining normal cellular function and liability. In the present study, we investigated the temporal immunohistochemical distribution of five representative components of NPCs-Ran GTPase-activating protein 1 (RanGap1), glycoprotein-210 (Gp210), nucleoporin 205 (Nup205), nucleoporin 107 (Nup107), and nucleoporin 50 (Nup50)-after 90 min of transient middle cerebral artery occlusion (tMCAO) up to 28 days after the reperfusion in rat brains. Single immunohistochemical analyses showed ring-like stainings along the periphery of the nucleus in sham control brains. After tMCAO, Gp210 and Nup107 immunoreactivity continuously increased from 1 day, and RanGap1, Nup205, and Nup50 increased from 2 days until 28 days, which also displayed progressive precipitations within the nucleus in the peri-ischemic area, while the ischemic core showed scarce expression with collapsed structure. Double immunofluorescent analyses revealed nuclear retention and apparent colocalization of RanGap1 with Nup205, Gp210 with Nup205, and partial colocalization of Nup205 with Nup107; most of the ischemic changes above were similar to those observed in patients with C9orf72-genetic amyotrophic lateral sclerosis. Taken together, these observations suggest that the mislocalization of these nucleoporins may be a common pathogenesis of both ischemic and neurodegenerative disease. © 2016 Wiley Periodicals, Inc.

Neural Stem Cell Transplantation Induces Stroke Recovery by Upregulating Glutamate Transporter GLT-1 in Astrocytes.

Tissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster axonal rewiring and dendritic plasticity and to induce long-term functional recovery. The observed therapeutic effect of neural precursor cells seems to underlie their capacity to upregulate the glial glutamate transporter on astrocytes through the vascular endothelial growth factor inducing favorable changes in the electrical and molecular stroke microenvironment. Cell-based approaches able to influence plasticity seem particularly suited to favor poststroke recovery.

Temporal Profiles of Stress Protein Inductions after Focal Transient Ischemia in Mice Brain

Stress proteins have been found to play important protective roles against ischemic brain injury under hypoxic, oxidative, heat shock, and proteasome stresses. In the present study, we investigated the temporal profiles of the major stress proteins including hypoxia-inducible factor-1α (HIF-1α), glutathione (GSH), heat shock protein 72 (HSP72), constitutive heat shock cognate protein 73 (HSC73), and ubiquitin after 45 minutes of transient middle cerebral artery occlusion (tMCAO) in the mice brain up to 7 days after reperfusion. Immunohistochemical analyses of HIF-1α, GSH, HSP72, and ubiquitin showed little immunoreactivity of neural cells in sham control brain, whereas HSC73 showed a constitutive immunoreactivity. After tMCAO, HSC73 showed the fastest increase at 12 hours in the peri-ischemic area, followed by HIF-1α with a peak at 24 hours, GSH, HSP72, and ubiquitin with a peak at 72 hours. All these stress proteins returned toward the baseline levels until 7 days. In the ischemic core, these stress proteins showed a similar change with less reaction compared to the peri-ischemic area. These data showed temporal expressions of HIF-1α, GSH, HSP72, HSC73, and ubiquitin in the mice brain after tMCAO, which might provide a better understanding of neuroprotective mechanisms and novel targets for therapeutic intervention of brain ischemic disease.

Anti-inflammatory effects of ADAMTS-4 in a mouse model of ischemic stroke.

ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs type 4) is a metalloprotease capable to degrade chondroitin sulfate proteoglycans leading to cartilage destruction during arthritis or to neuroplasticity during spinal cord injury (SCI). Although ADAMTS-4 is an inflammatory-regulated enzyme, its role during inflammation has never been investigated. The aim of this study was to investigate the role of ADAMTS-4 in neuroinflammation. First, we evidenced an increase of ADAMTS-4 expression in the ischemic brain hemisphere of mouse and human patients suffering from ischemic stroke. Then, we described that ADAMTS-4 has predominantly an anti-inflammatory effect in the CNS. Treatment of primary microglia or astrocyte cultures with low doses of a human recombinant ADAMTS-4 prior to LPS exposure decreased NO production and the synthesis/release of pro-inflammatory cytokines including NOS2, CCL2, TNF-α, IL-1β and MMP-9. Accordingly, when cell cultures were transfected with silencing siRNA targeting ADAMTS-4 prior to LPS exposure, the production of NO and the synthesis/release of pro-inflammatory cytokines were increased. Finally, the feasibility of ADAMTS-4 to modulate neuroinflammation was investigated in vivo after permanent middle cerebral artery occlusion in mice. Although ADAMTS-4 treatment did not influence the lesion volume, it decreased astrogliosis and macrophage infiltration, and increased the number of microglia expressing arginase-1, a marker of alternatively activated cells with inflammation inhibiting functions. Additionally, ADAMTS-4 increased the production of IL-10 and IL-6 in the peri-ischemic area. By having anti-inflammatory and neuroregenerative roles, ADAMTS-4 may represent an interesting target to treat acute CNS injuries, such as ischemic stroke, SCI or traumatic brain injury. GLIA 2016;64:1492-1507.

Immunomodulation by interleukin-33 is protective in stroke through modulation of inflammation.

Cerebral stroke induces massive Th1-shifted inflammation both in the brain and the periphery, contributing to the outcome of stroke. A Th1-type response is neurotoxic whereas a Th2-type response is accompanied by secretion of anti-inflammatory cytokines, such as interleukin-4 (IL-4). Interleukin-33 (IL-33) is a cytokine known to induce a shift towards the Th2-type immune response, polarize macrophages/microglia towards the M2-type, and induce production of anti-inflammatory cytokines. We found that the plasma levels of the inhibitory IL-33 receptor, sST2, are increased in human stroke and correlate with a worsened stroke outcome, suggesting an insufficient IL-33-driven Th2-type response. In mouse, peripheral administration of IL-33 reduced stroke-induced cell death and improved the sensitivity of the contralateral front paw at 5days post injury. The IL-33-treated mice had increased levels of IL-4 in the spleen and in the peri-ischemic area of the cortex. Neutralization of IL-4 by administration of an IL-4 antibody partially prevented the IL-33-mediated protection. IL-33 treatment also reduced astrocytic activation in the peri-ischemic area and increased the number of Arginase-1 immunopositive microglia/macrophages at the lesion site. In human T-cells, IL-33 treatment induced IL-4 secretion, and the conditioned media from IL-33-exposed T-cells reduced astrocytic activation. This study demonstrates that IL-33 is protective against ischemic insult by induction of IL-4 secretion and may represent a novel therapeutic approach for the treatment of stroke.

12 hours after cerebral ischemia is the optimal time for bone marrow mesenchymal stem cell transplantation.

Cell therapy using stem cell transplantation against cerebral ischemia has been reported. However, it remains controversial regarding the optimal time for cell transplantation and the transplantation route. Rat models of cerebral ischemia were established by occlusion of the middle cerebral artery. At 1, 12 hours, 1, 3, 5 and 7 days after cerebral ischemia, bone marrow mesenchymal stem cells were injected via the tail vein. At 28 days after cerebral ischemia, rat neurological function was evaluated using a 6-point grading scale and the pathological change of ischemic cerebral tissue was observed by hematoxylin-eosin staining. Under the fluorescence microscope, the migration of bone marrow mesenchymal stem cells was examined by PKH labeling. Caspase-3 activity was measured using spectrophotometry. The optimal neurological function recovery, lowest degree of ischemic cerebral damage, greatest number of bone marrow mesenchymal stem cells migrating to peri-ischemic area, and lowest caspase-3 activity in the ischemic cerebral tissue were observed in rats that underwent bone marrow mesenchymal stem cell transplantation at 12 hours after cerebral ischemia. These findings suggest that 12 hours after cerebral ischemia is the optimal time for tail vein injection of bone marrow mesenchymal stem cell transplantation against cerebral ischemia, and the strongest neuroprotective effect of this cell therapy appears at this time.

Improvement of Cardiac Stem Cell Sheet Therapy for Chronic Ischemic Injury by Adding Endothelial Progenitor Cell Transplantation: Analysis of Layer-Specific Regional Cardiac Function

The transplantation of cardiac stem cell sheets (CSC sheets) is a promising therapeutic strategy for ischemic cardiomyopathy, although potential ischemia in the transplanted area remains a problem. Injected endothelial progenitor cells (EPCs) can reportedly induce angiogenesis in the injected area. We hypothesized that concomitant CSC sheet transplantation and EPC injection might show better therapeutic effects for chronic ischemic injury model than the transplantation of CSC sheets alone. Scaffold-free CSC sheets were generated from human c-kit-positive heart-derived cells. A porcine chronic ischemic injury model was generated by placing an ameroid constrictor around the left coronary artery for 4 weeks. The animals then underwent a sham operation, epicardial transplantation of CSC sheet over the ischemic area, intramyocardial injection of EPCs into the ischemic and peri-ischemic area, or CSC sheet transplantation plus EPC injection. The efficacy of each treatment was then assessed for 2 months. Speckle-tracking echocardiography was used to dissect the layer-specific regional systolic function by measuring the radial strain (RS). The epicardial RS in the ischemic area was similarly greater after treatment with the CSC-derived cell sheets alone (19 ± 5%) or in combination with EPC injection (20 ± 5%) compared with the EPC only (9 ± 4%) or sham (7 ± 1%) treatment. The endocardial RS in the ischemic area was greatest after the combined treatment (14 ± 1%), followed by EPC only (12 ± 1%), compared to the CSC only (11 ± 1%) and sham (9 ± 1%) treatments. Consistently, either epicardial CSC sheet implantation or intramyocardial EPC injection yielded increased capillary number and reduced cardiac fibrosis in the ischemic epicardium or endocardium, respectively. Concomitant EPC injection induced the migration of transplanted CSCs into the host myocardium, leading to further neovascularization and reduced fibrosis in the ischemic endocardium, compared to the CSC sole therapy. Transplantation of CSC sheets induced significant functional recovery of the ischemic epicardium, and concomitant EPC transplantation elicited transmural improvement in chronic ischemic injury.

Anti-salusin-β antibody enhances angiogenesis after myocardial ischemia reperfusion injury

Backgrounds: Salusins are multifunctional endogenous bioactive peptides simultaneously biosynthesized from their precursor prosalusin. Salusin-β stimulates proliferation of vascular smooth muscle cells and fibroblasts and regulates myocardial growth and hypertrophy. Salusin-β has potent hypotensive, bradycardic and proatherosclerotic effects.Objectives: To investigate whether salusin-β plays a role in myocardial remodeling after myocardial ischemia reperfusion (I/R) injury, rat I/R models were created by the left anterior descending coronary artery occlusion for 30 min, followed by 24 h or 7 days of reperfusion (control, n = 6 each).Results and Conclusion: Immunohistochemical double staining showed the enhanced expression of salusin-β in the macrophages around myocardial ischemic area. Anti-salusin-β treated groups were administered the neutralizing salusin-β antibody (10 μl/day, i.p.) once daily from day −1 to day 1 or from day −1 to day 7 (anti-salusin-β, n = 6 each). The anti-salusin-β therapy enhanced myocardial angiogenesis in the peri-ischemic area of reperfusion. The small vessels (< 40 μm in diameter) of I/R hearts treated with anti-salusin-β were more densely populated than those of control animals (108.5 ± 19.7 vs 47.5 ± 2.4, p < 0.05). Real-time PCR revealed that the anti-salusin-β therapy-induced angiogenesis was not associated with enhanced vascular endothelial growth factor A expression. The authors, for the first time, have clarified that endogenous salusin-β suppresses angiogenesis which is critical in the development of cardiac remodeling following I/R injury.

The Effect of Continuous Epidural Electrical Stimulation on Neuronal Proliferation in Cerebral Ischemic Rats

ObjectiveTo investigate the effect of electrical stimulation (ES) on the recovery of motor skill and neuronal cell proliferation.MethodsThe male Sprague-Dawley rats were implanted with an epidural electrode over the peri-ischemic area after photothrombotic stroke in the dominant sensorimotor cortex. All rats were randomly assigned into the ES group and control group. The behavioral test of a single pellet reaching task (SPRT) and neurological examinations including the Schabitz's photothrombotic neurological score and the Menzies test were conducted for 2 weeks. After 14 days, coronal sections were obtained and immunostained for neuronal cell differentiation markers including bromodeoxyuridine (BrdU), neuron-specific nuclear protein (NeuN), and doublecortin (DCX).ResultsOn the SPRT, the motor function in paralytic forelimbs of the ES group was significantly improved. There were no significant differences in neurological examinations and neuronal cell differentiation markers except for the significantly increased number of DCX+ cells in the corpus callosum of the ES group (p<0.05). But in the ES group, the number of NeuN+ cells in the ischemic cortex and the number of NeuN+ cells and DCX+ cells in the ischemic striatum tended to increase. In the ES group, NeuN+ cells in the ischemic hemisphere and DCX+ cells and BrdU+ cells in the opposite hemisphere tended to increase compared to those in the contralateral.ConclusionThe continuous epidural ES of the ischemic sensorimotor cortex induced a significant improvement in the motor function and tended to increase neural cell proliferation in the ischemic hemisphere and the neural regeneration in the opposite hemisphere.