Peri-infarct Area Research Articles

Cardiac sympathetic afferent denervation reduces matrix metalloproteinase expression and improves cardiac remodeling in rats post myocardial infarction

Abstract In a previous study from our laboratory, we demonstrated that chronic and selective cardiac sympathetic afferent (spinal) denervation at the time of myocardial infarction (MI) using epicardial application of resiniferatoxin (RTX), a neuronal toxin capable of inducing rapid degeneration of transient receptor potential vanilloid 1 (TRPV1)-expressing afferent neurons and fibers, markedly reduced the cardiac remodeling process 9–11 weeks post-MI in rats. This included reduced cardiac hypertrophy, fibrosis and apoptosis. Here, we further investigated the effect of epicardial application of RTX at the time of MI on cardiac extracellular matrix (ECM) and remodeling post-MI. Echocardiographic and morphologic data demonstrated that, compared to MI+vehicle, MI+RTX exhibited a significantly slower LV chamber dilation (6-week echocardiographic data: left ventricular end-diastolic diameter, 10.7±0.2 vs. 9.6±0.3 mm; left ventricular end-systolic diameter: 8.8±0.2 vs. 7.8±0.3 mm; MI+vehicle vs. MI+RTX, n=18, p<0.05; mean±SE). Scanning electron microscopy showed that RTX reduced collagen deposition in the peri-infarct area in post-MI rats. Western blot and zymography were used to further evaluate the effect of RTX application on matrix metalloproteinase (MMP) expression and activity, which is responsible for degrading ECM and contributing to cardiac dilation post MI. Our data suggest time-dependent increases in MMP expression in infarcted hearts post MI. RTX application largely prevented the increase in MMP9 but not MMP2, 4 weeks post-MI. RTX prevented both increased MMP2 and MMP9 activities in the peri-infarct myocardium at 8–10 weeks post-MI. We further investigated the effect of RTX application on MI-induced cardiac inflammation, which has been reported as an upstream mechanism triggering MMP activation post-MI. The data show that RTX largely abolished MI-induced plasma extravasation and reduced macrophage infiltration and cardiac cytokine content in the peri-infarct or remote myocardium of post-MI rats. These data suggest that cardiac sympathetic afferent denervation at the time of MI exerts a local anti-inflammatory effect and reduces ECM remodeling by preventing excessive MMP activation in post-MI rats. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institutes of Health Grant HL126796

FAIM Enhances the Efficacy of Mesenchymal Stem Cell Transplantation by Inhibiting JNK-Induced c-FLIP Ubiquitination and Degradation.

Background The poor survival rates of transplanted mesenchymal stem cells (MSCs) in harsh microenvironments impair the efficacy of MSCs transplantation in myocardial infarction (MI). Extrinsic apoptosis pathways play an important role in the apoptosis of transplanted MSCs, and Fas apoptosis inhibitory molecule (FAIM) is involved in regulation of the extrinsic apoptosis pathway. Thus, we aimed to explore whether FAIM augmentation protects MSCs against stress-induced apoptosis and thereby improves the therapeutic efficacy of MSCs. Methods We ligated the left anterior descending coronary artery (LAD) in the mouse heart to generate an MI model and then injected FAIM-overexpressing MSCs (MSCsFAIM) into the peri-infarction area in vivo. Moreover, FAIM-overexpressing MSCs were challenged with oxygen, serum, and glucose deprivation (OGD) in vitro, which mimicked the harsh microenvironment that occurs in cardiac infarction. Results FAIM was markedly downregulated under OGD conditions, and FAIM overexpression protected MSCs against OGD-induced apoptosis. MSCsFAIM transplantation improved cell retention, strengthened angiogenesis, and ameliorated heart function. The antiapoptotic effect of FAIM was mediated by cellular-FLICE inhibitory protein (c-FLIP), and FAIM augmentation improved the protein expression of c-FLIP by reducing ubiquitin–proteasome-dependent c-FLIP degradation. Furthermore, FAIM inhibited the activation of JNK, and treatment with the JNK inhibitor SP600125 abrogated the reduction in c-FLIP protein expression caused by FAIM silencing. Conclusions Overall, these results indicated that FAIM curbed the JNK-mediated, ubiquitination–proteasome-dependent degradation of c-FLIP, thereby improving the survival of transplanted MSCs and enhancing their efficacy in MI. This study may provide a novel approach to strengthen the therapeutic effect of MSC-based therapy.

Limb-Bud and Heart (LBH) Upregulation in Cardiomyocytes under Hypoxia Promotes the Activation of Cardiac Fibroblasts via Exosome Secretion.

The activation of cardiac fibroblasts (CFs) after myocardial infarction (MI) is essential for post-MI infarct healing, during which the regulation of transforming growth factor beta1 (TGF-β1) signaling is predominant. We have demonstrated that TGF-β1-mediated upregulation of LBH contributes to post-MI CF activation via modulating αB-crystallin (CRYAB), after being upregulated by TGF-β1. In this study, the effect of LBH-CRYAB signaling on the cardiac microenvironment via exosome communication and the corresponding mechanisms were investigated. The upregulation of LBH and CRYAB was verified in both cardiomyocytes (CMs) and CFs in hypoxic, post-MI peri-infarct tissues. CM-derived exosomes were isolated and identified, and LBH distribution was elevated in exosomes derived from LBH-upregulated CMs under hypoxia. Treatment with LBH+ exosomes promoted cellular proliferation, differentiation, and epithelial-mesenchymal transition-like processes in CFs. Additionally, in primary LBHKO CFs, western blotting showed that LBH knockout partially inhibited TGF-β1-induced CF activation, while LBH-CRYAB signaling affected TGF-β1 expression and secretion through a positive feedback loop. The administration of a Smad3 phosphorylation inhibitor to LBHKO CFs under TGF-β1 stimulation indicated that Smad3 phosphorylation partially accounted for TGF-β1-induced LBH upregulation. In conclusion, LBH upregulation in CMs in post-MI peri-infarct areas correlated with a hypoxic cardiac microenvironment and led to elevated exosomal LBH levels, promoting the activation of recipient CFs, which brings new insights into the studies and therapeutic strategies of post-MI cardiac repair.

Sympathetic activation in heart failure.

Sympathetic activation has been long appreciated exclusively as a fundamental compensatory mechanism of the failing heart and, thus, welcome and to be supported. In the initial clinical phases of heart failure (HF), the sympathetic nervous system overdrive plays a compensatory function aimed at maintaining an adequate cardiac output despite the inotropic dysfunction affecting the myocardium. However, when the sympathetic reflex response is exaggerated it triggers a sequence of unfavourable remodelling processes causing a further contractile deterioration that unleashes major adverse cardiovascular consequences, favouring the HF progression and the occurrence of fatal events. Eventually, the sympathetic nervous system in HF was demonstrated to be a ‘lethality factor’ and thus became a prominent therapeutic target. The existence of an effective highly specialized intracardiac neuronal network immediately rules out the old concept that sympathetic activation in HF is merely the consequence of a drop in cardiac output. When a cardiac damage occurs, such as myocardial ischaemia or a primary myocardial disorder, the adaptive capability of the system may be overcame, leading to excessive sympatho-excitation coupled with attenuation till to abolishment of central parasympathetic drive. Myocardial infarction causes, within a very short time, both a functional and anatomical remodelling with a diffuse up-regulation of nerve growth factor (NGF). The subsequent nerve sprouting signal, facilitated by a rise in the levels of NGF in the left stellate ganglion and in the serum, triggers an increase in cardiac nerve density in both peri-infarct and non-infarcted areas. Finally, NFG production decreases over time, supposedly as an adaptative response to the prolonged exposure to sympathetic overactivity, leading in the end to a reduction in sympathetic nerve density. Accordingly, NGF levels were markedly reduced in patients with severe congestive heart failure. The kidney is the other key player of the sympathetic response to HF as it indeed reacts to under-perfusion and to loop diuretics to preserve filtration at the cost of many pathological consequences on its physiology. This vicious loop ultimately participates to the chronic and disruptive sympathetic overdrive.In conclusion, sympathetic activation is the natural physiological consequence to life stressors but also to any condition that may harm our body. It is the first system of reaction to any potential life-threatening event. However, in any aspect of life over reaction is never effective but, in many instances, is, actually, life threatening. One for all is the case of ischaemia-related ventricular fibrillation which is, strongly facilitated by sympathetic hyperactivity. The take home message? When, in a condition of harm, everybody is yelling failure is just around the corner.

Purpurogallin improves neurological functions of cerebral ischemia and reperfusion mice by inhibiting endoplasmic reticulum stress and neuroinflammation

BackgroundPurpurogallin (PPG) has been testified to have neuroprotective effects. This study intends to probe the neuroprotection of PPG on cerebral ischemia/reperfusion (I/R) injury and its potential mechanism. MethodsC57/B6 mice, BV2 microglia and HT22 hippocampal neurons were used for in-vivo and in-vitro experiments. I/R injury models were constructed using middle cerebral artery occlusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R), respectively. The expression of apoptosis and inflammatory proteins, and endoplasmic reticulum (ER) stress proteins were gauged by Western blotting (WB). The contents of inflammatory cytokines in OGD/R-induced BV2 microglia were testified by enzyme-linked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8), TUNEL assay and flow cytometry (FCM) were utilized to examine the viability and apoptosis of cells. The neurological, learning and memory functions were evaluated by the modified neurological severity score (mNSS) and water maze experiment. 2, 3, 5-triphenyltetrazole chloride (TTC) staining was utilized to calculate the volume of cerebral infarction and cerebral edema in the peri-infarct area. Apoptosis-related proteins, inflammation-related proteins and ER stress proteins were gauged by WB. ELISA was conducted to verify inflammatory cytokines. ResultsPPG treatment notably abated the expression of ER stress proteins and inflammatory factors in OGD/R-induced BV2 microglia and boosted HT22 neuron's viability and eased their apoptosis in comparison to the control group. In vivo, PPG treatment signally lessened cerebral infarct area, cerebral edema, and neurological deficit scores in MCAO/R mice. Additionally, PPG caused a dramatic decline in neuronal apoptosis and levels of ER stress proteins and inflammatory factors in the brain’s peri-infarct region of MCAO/R mice. Mechanically, PPG blocked the TLR4/NF-κB pathway in OGD/R-induced BV2, HT22 neurons, and the MCAO/R mice. ConclusionPPG attenuates brain I/R damage probably by suppressing ER stress and neuroinflammation via inactivation of the TLR4/NF-κB pathway, suggesting that PPG may be a candidate drug for treating cerebral I/R injury.

Sex differences in cardiac remodeling post myocardial infarction with acute cigarette smoking

BackgroundWhether cigarette smoking affects the heart post-myocardial infarction (MI) in a sex-dependent way remains controversial. Using a mouse model, we investigated cardiac remodeling under the influence of acute cigarette smoke (CS) exposure following ischemic injury in both sexes.MethodsTen cigarettes were smoked twice daily for 2 weeks followed by MI and then 1 additional week post permanent LAD ligation. Cardiac function, histology, and infarct size were assessed, and inflammatory markers quantified by RT–PCR. Statistical comparisons were performed using an unpaired t test or ANOVA followed by Tukey post hoc test.ResultsWe observed that cigarette smoking exacerbated both left and right ventricular remodeling only in males at an early stage of post-MI. Females did not display a significant structural and/or functional alteration within 7 days of cardiac remodeling post-MI upon CS exposure. Worsened right ventricular remodeling in males was independent of pulmonary congestion. CS-exposed males exhibited enhanced increases in left ventricular end systolic and diastolic volumes, as well as reductions in ejection fraction and fractional area changes of left ventricular base. At day 7, infarct size was increased by cigarette smoking in males only, which was accompanied by enhanced collagen deposition in both the infarcted and peri-infarcted areas. Both IL-6 and TNF-α mRNA expression significantly increased in CS-exposed MI male group only at day 7 post-MI suggestive of prolonged inflammation.ConclusionsThese findings indicate that CS exposure worsens the progression of cardiac remodeling post-MI in male sex in a significant manner compared to female sex at least at early stages.

Rab27a-dependent exosomes protect against cerebral ischemic injury by reducing endothelial oxidative stress and apoptosis.

IntroductionMulticellular crosstalk within the brain tissue has been suggested to play a critical role in maintaining cerebral vascular homeostasis. Exosomes (EXs) mediated cell–cell communication, but its role in cerebral ischemic injury is largely unknown. Rab27a is one of the major genes controlling EX release. Here, we explored the role of Rab27a in regulating brain EXs secretion, and the effects of Rab27a‐mediated EXs on ischemia evoked cerebral vascular disruption and brain injury.MethodsCerebral ischemia was induced in Rab27a knockout (Rab27a−/−) and wide type (WT) mice by transient middle cerebral artery occlusion (tMCAO). Differential gene expression analysis was performed in ischemic brain tissue by using mRNA sequencing. EXs isolated from brain tissue of Rab27a−/− and WT mice (EXWT or EXRab27a−/−) were pre‐administrated into tMCAO operated Rab27a−/− mice or oxygen and glucose deprivation (OGD) treated primary brain vascular endothelial cells (ECs).ResultsWe demonstrated that Rab27a expression in the peri‐infarct area of brain was significantly elevated, which was associated with local elevation in EXs secretion. Rab27a deficiency dramatically decreased the level of EXs in brain tissue of normal and tMCAO‐treated mice, and Rab27a−/− mice displayed an increase in infarct volume and NDS, and a decrease in cMVD and CBF following tMCAO. Pre‐infusion of EXWT increased the brain EXs levels in the tMCAO operated Rab27a−/− mice, accompanied with an increase in cMVD and CBF, and a decrease in infarct volume, NDS, ROS production, and apoptosis. The effects of EXRab27a−/− infusion were much diminished although in a dose‐dependent manner. In OGD‐treated ECs, EXRab27a−/− showed less effectivity than EXWT in decreasing ROS overproduction and apoptosis, paralleling with down‐regulated expression of NOX2 and cleaved caspase‐3.ConclusionOur study demonstrates that Rab27a controls brain EXs secretion and functions, contributing to cerebral vascular protection from ischemic insult by preventing oxidative stress and apoptosis via down‐regulating NOX2 and cleaved caspase‐3 expression.

Pregabalin improves axon regeneration and motor outcome in a rodent stroke model.

Ischaemic stroke remains a leading cause of death and disability worldwide. Surviving neurons in the peri-infarct area are able to establish novel axonal projections to juxtalesional regions, but this regeneration is curtailed by a growth-inhibitory environment induced by cells such as reactive astrocytes in the glial scar. Here, we found that the astroglial synaptogenic cue thrombospondin-1 is upregulated in the peri-infarct area, and hence tested the effects of the anticonvulsant pregabalin, a blocker of the neuronal thrombospondin-1 receptor Alpha2delta1/2, in a mouse model of cortical stroke. Studying axonal projections after cortical stroke in mice by three-dimensional imaging of cleared whole-brain preparations, we found that pregabalin, when administered systemically for 5 weeks after stroke, augments novel peri-infarct motor cortex projections and improves skilled forelimb motor function. Thus, the promotion of axon elongation across the glial scar by pregabalin represents a promising target beyond the acute phase after stroke to improve structural and functional recovery.

Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NFκB/NLRP3 signaling pathway in cerebral ischemic mice

BackgroundNeuronal pyroptosis and neuroinflammation with excess microglial activation are widely involved in the early pathological process of ischemic stroke. Repetitive transcranial magnetic stimulation (rTMS), as a non-invasive neuromodulatory technique, has recently been reported to be anti-inflammatory and regulate microglial function. However, few studies have elucidated the role and mechanism of rTMS underlying regulating neuronal pyroptosis and microglial polarization.MethodsWe evaluated the motor function in middle cerebral artery occlusion/reperfusion (MCAO/r) injury mice after 1-week intermittent theta-burst rTMS (iTBS) treatment in the early phase with or without depletion of microglia by colony-stimulating factor 1 receptor (CSF1R) inhibitor treatment, respectively. We further explored the morphological and molecular biological alterations associated with neuronal pyroptosis and microglial polarization via Nissl, EdU, TTC, TUNEL staining, electron microscopy, multiplex cytokine bioassays, western blot assays, immunofluorescence staining and RNA sequencing.ResultsITBS significantly protected against cerebral ischemia/reperfusion (I/R) injury-induced locomotor deficits and neuronal damage, which probably relied on the regulation of innate immune and inflammatory responses, as evidenced by RNA sequencing analysis. The peak of pyroptosis was confirmed to be later than that of apoptosis during the early phase of stroke, and pyroptosis was mainly located and more severe in the peri-infarcted area compared with apoptosis. Multiplex cytokine bioassays showed that iTBS significantly ameliorated the high levels of IL-1β, IL-17A, TNF-α, IFN-γ in MCAO/r group and elevated the level of IL-10. ITBS inhibited the expression of neuronal pyroptosis-associated proteins (i.e., Caspase1, IL-1β, IL-18, ASC, GSDMD, NLRP1) in the peri-infarcted area rather than at the border of infarcted core. KEGG enrichment analysis and further studies demonstrated that iTBS significantly shifted the microglial M1/M2 phenotype balance by curbing proinflammatory M1 activation (Iba1+/CD86+) and enhancing the anti-inflammatory M2 activation (Iba1+/CD206+) in peri-infarcted area via inhibiting TLR4/NFκB/NLRP3 signaling pathway. Depletion of microglia using CSF1R inhibitor (PLX3397) eliminated the motor functional improvements after iTBS treatment.ConclusionsrTMS could alleviate cerebral I/R injury induced locomotor deficits and neuronal pyroptosis by modulating the microglial polarization. It is expected that these data will provide novel insights into the mechanisms of rTMS protecting against cerebral I/R injury and potential targets underlying neuronal pyroptosis in the early phase of stroke.

Beclin1 haploinsufficiency compromises mesenchymal stem cell-offered cardioprotection against myocardial infarction

Mesenchymal stem cells (MSCs)-based therapy has displayed some promises in ischemia heart diseases although its efficacy may be affected by changes in surrounding environments. This study evaluated the role of autophagy insufficiency using Beclin1 haploinsufficiency (BECN+/−) on intra-myocardial MSC transplantation-evoked effect against myocardial infarction. Donor MSCs from C57BL/6 mice were labelled with cell-tracker CM Dil and were delivered into LV free wall adjacent to infarct region in wild-type (WT) and BECN+/− recipient mice following ligation of left main coronary artery (MI-MSCs). Ten days following MI, myocardial function was assessed using echocardiography. Cardiomyocyte contractility and intracellular Ca2+ were monitored using cardiomyocytes from the area-at-risk adjacent to infarct. CM-Dil labeled cells were tracked in MSCs recipient mice using fluorescence microscopy. Lectin, Masson trichrome staining and Western blot analysis were employed to determine cardiomyocyte area, scar fibrosis, apoptosis and inflammation. MI insult triggered scar fibrosis, LV chamber dilation, decreased fractional shortening, ejection fraction, cardiomyocyte shortening, maximal velocity of shortening and relengthening as well as prolonged relengthening, which were abrogated or attenuated by MSCs therapy in WT but not BECN+/− mice. MI decreased intracellular Ca2+ rise and decay in response to electrical stimuli without affecting resting intracellular Ca2+, which were reconciled by MSCs in WT but not BECN+/− mice. MSCs further attenuated MI-induced mitochondrial ultrastructural injury, apoptosis, inflammation and autophagy defects in peri-infarct area in WT but not BECN+/− mice. Collectively, our results suggested that autophagy insufficiency dampened in MSCs-elicited cardioprotection associated with dampened apoptosis and inflammation.

Behind the Wall-Compartment-Specific Neovascularisation during Post-Stroke Recovery in Mice.

Ischemic stroke is a highly prevalent vascular disease leading to oxygen- and glucose deprivation in the brain. In response, ischemia-induced neovascularization occurs, which is supported by circulating CD34+ endothelial progenitor cells. Here, we used the transient middle cerebral artery occlusion (tMCAO) mouse model to characterize the spatio-temporal alterations within the ischemic core from the acute to the chronic phase using multiple-epitope-ligand cartography (MELC) for sequential immunohistochemistry. We found that around 14 days post-stroke, significant angiogenesis occurs in the ischemic core, as determined by the presence of CD31+/CD34+ double-positive endothelial cells. This neovascularization was accompanied by the recruitment of CD4+ T-cells and dendritic cells as well as IBA1+ and IBA1− microglia. Neighborhood analysis identified, besides pericytes only for T-cells and dendritic cells, a statistically significant distribution as direct neighbors of CD31+/CD34+ endothelial cells, suggesting a role for these cells in aiding angiogenesis. This process was distinct from neovascularization of the peri-infarct area as it was separated by a broad astroglial scar. At day 28 post-stroke, the scar had emerged towards the cortical periphery, which seems to give rise to a neuronal regeneration within the peri-infarct area. Meanwhile, the ischemic core has condensed to a highly vascularized subpial region adjacent to the leptomeningeal compartment. In conclusion, in the course of chronic post-stroke regeneration, the astroglial scar serves as a seal between two immunologically active compartments—the peri-infarct area and the ischemic core—which exhibit distinct processes of neovascularization as a central feature of post-stroke tissue remodeling. Based on our findings, we propose that neovascularization of the ischemic core comprises arteriogenesis as well as angiogenesis originating from the leptomenigeal vasculature.

Chronic Vagal Nerve Stimulation Rescues Sympathetic Control of the Heart following Myocardial Infarction

Cardiac injury, such as myocardial infarction (MI), leads to neurohormonal activation and autonomic remodeling, resulting in reflexive sympathoexcitation and parasympathetic dysfunction, which further contribute to progression of disease. The purpose of this study was to determine whether chronic cervical vagal nerve stimulation (VNS) influences MI‐associated changes in cardiac sympathetic function. Yucatan minipigs were divided into control (n=6), chronic MI (n=7), and chronic MI + chronic VNS (n=8) groups. VNS therapy was delivered to the right cervical vagus nerve and systematically titrated to an optimal intensity based on heart rate dynamics (5Hz, 2.1±0.3mA, 250μs, 17.5% duty cycle) using telemetry in the conscious state. Chronic MI was created using percutaneous embolization of the left anterior descending coronary artery with polystyrene beads under fluoroscopy (Figure 1A). VNS therapy was started 2 days following MI, and the animals maintained for 6‐8 weeks. At terminal study, we evaluated hemodynamic responses to graded bilateral sympathetic chain stimulation (BSS). Left ventricular (LV) strips encompassing regions of scar, border zone, and normal myocardium were fixed in 10% formalin, embedded in paraffin, sectioned, and studied using Masson's trichrome stain (Figure 1B‐C). Structural changes at the peri‐infarct area were scored (0‐3) for myocyte morphology and presence of hypertrophy or myocytolysis. Animals with MI alone exhibited spontaneous ventricular arrhythmias, including two that experienced sudden cardiac death (Figure 1D). At terminal study, there were no significant differences in baseline systolic function among groups. Moderate (4Hz) and high (10Hz) intensity BSS increased LV contractility (dp/dtmax) in all groups (Figure 2A‐B); however, evoked changes were significantly reduced in the chronic MI compared to control or MI + VNS groups. While control animals displayed evidence of contractile reserve at 10Hz vs 4Hz (1297±266 mmHg/s, p=0.03), chronic MI animals did not (631.9±264 mmHg/s, p=0.11), which was improved with chronic VNS therapy (1027±160.6 mmHg/s, p<0.01, Figure 2C). Chronic VNS therapy also significantly reduced MI‐associated structural remodeling in the peri‐infarct area, including the degree of myocytolysis and dysmorphic myocytes (structural abnormality index 0.6±0.2 vs 1.8±0.2, p<0.01). Our findings suggest that chronic VNS ameliorates sympathetic functional remodeling following myocardial infarction and improves cardiac mechanical performance in response to simulated stress. Furthermore, chronic VNS reduces pathologic myocardial remodeling at the peri‐infarct zone, potentially stabilizing the resultant ventricular scar.

Intravital Imaging Reveals the Ameliorating Effect of Colchicine in a Photothrombotic Stroke Model via Inhibition of Neutrophil Recruitment.

Although post-stroke neutrophil recruitment is known to be deleterious to neural tissues in the peri-infarct area, the precise behavior of recruited neutrophils remains elusive. In this study, potential therapeutic agents for modifying neutrophil behavior in the peri-infarct area were explored through intravital imaging of an experimental stroke mouse model. By applying in vivo 2-photon imaging to study a tightly controlled photothrombotic stroke mouse model, we established a highly sensitive and reproducible method for investigating the temporal dynamics of ischemic brain lesions. Taking advantage of this system, we revealed that neutrophil depletion by a neutrophil-specific antibody ameliorated the expansion of the infarct area, confirming the deleterious effect of neutrophils in the peri-infarct cortex. To identify neutrophil-targeted therapeutic approaches, we screened various agents and found that colchicine and an anti-P-selectin antibody were the most effective in inhibiting neutrophil attachment to the vessel wall in the early phase (6h post-infarction). Interestingly, further investigation in the later phase (16h post-infarction) revealed that colchicine potently inhibited neutrophil infiltration into the peri-infarct cortex; however, the anti-P-selectin antibody did not. Subsequent analysis revealed that the effect of the anti-P-selectin antibody against neutrophil attachment to the vessel wall was transient and thus insufficient for mitigating neutrophil infiltration. Finally, we revealed that colchicine treatment effectively ameliorated infarct expansion. In conclusion, we have established an intravital strategy to directly investigate pathophysiology in the ischemic border zone, and found that colchicine administration in the acute phase of ischemic stroke is a potential novel therapeutic strategy.

Growth Hormone Increases BDNF and mTOR Expression in Specific Brain Regions after Photothrombotic Stroke in Mice.

Aims We have shown that growth hormone (GH) treatment poststroke increases neuroplasticity in peri-infarct areas and the hippocampus, improving motor and cognitive outcomes. We aimed to explore the mechanisms of GH treatment by investigating how GH modulates pathways known to induce neuroplasticity, focusing on association between brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) in the peri-infarct area, hippocampus, and thalamus. Methods Recombinant human growth hormone (r-hGH) or saline was delivered (0.25 μl/hr, 0.04 mg/day) to mice for 28 days, commencing 48 hours after photothrombotic stroke. Protein levels of pro-BDNF, total-mTOR, phosphorylated-mTOR, total-p70S6K, and phosporylated-p70S6K within the peri-infarct area, hippocampus, and thalamus were evaluated by western blotting at 30 days poststroke. Results r-hGH treatment significantly increased pro-BDNF in peri-infarct area, hippocampus, and thalamus (p < 0.01). r-hGH treatment significantly increased expression levels of total-mTOR in the peri-infarct area and thalamus (p < 0.05). r-hGH treatment significantly increased expression of total-p70S6K in the hippocampus (p < 0.05). Conclusion r-hGH increases pro-BDNF within the peri-infarct area and regions that are known to experience secondary neurodegeneration after stroke. Upregulation of total-mTOR protein expression in the peri-infarct and thalamus suggests that this might be a pathway that is involved in the neurorestorative effects previously reported in these animals and warrants further investigation. These findings suggest region-specific mechanisms of action of GH treatment and provide further understanding for how GH treatment promotes neurorestorative effects after stroke.

Microanatomical study of pyramidal neurons in the contralesional somatosensory cortex after experimental ischemic stroke.

At present, many studies support the notion that after stroke, remote regions connected to the infarcted area are also affected and may contribute to functional outcome. In the present study, we have analyzed possible microanatomical alterations in pyramidal neurons from the contralesional hemisphere after induced stroke. We performed intracellular injections of Lucifer yellow in pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere in an ischemic stroke mouse model. A detailed 3-dimensional analysis of the neuronal complexity and morphological alterations of dendritic spines was then performed. Our results demonstrate that pyramidal neurons from layer III in the somatosensory cortex of the contralesional hemisphere show selective changes in their dendritic arbors, namely, less dendritic complexity of the apical dendritic arbor-but no changes in the basal dendritic arbor. In addition, we found differences in spine morphology in both apical and basal dendrites comparing the contralesional hemisphere with the lesional hemisphere. Our results show that pyramidal neurons of remote areas connected to the infarct zone exhibit a series of selective changes in neuronal complexity and morphological distribution of dendritic spines, supporting the hypothesis that remote regions connected to the peri-infarcted area are also affected after stroke.

1α,25-Dihydroxyvitamin D3 Promotes Angiogenesis After Cerebral Ischemia Injury in Rats by Upregulating the TGF-β/Smad2/3 Signaling Pathway.

Stroke is a disease with high morbidity, disability and mortality, which seriously endangers the life span and quality of life of people worldwide. Angiogenesis and neuroprotection are the key to the functional recovery of penumbra function after acute cerebral infarction. In this study, we used the middle cerebral artery occlusion (MCAO) model to investigate the effects of 1α,25-dihydroxyvitamin D3 (1,25-D3) on transforming growth factor-β (TGF-β)/Smad2/3 signaling pathway. Cerebral infarct volume was measured by TTC staining. A laser speckle flow imaging system was used to measure cerebral blood flow (CBF) around the ischemic cortex of the infarction, followed by platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and isolectin-B4 (IB4) immunofluorescence. The expression of vitamin D receptor (VDR), TGF-β, Smad2/3, p-Smad2, p-Smad3, and vascular endothelial growth factor (VEGF) was analyzed by western blot and RT-qPCR. Results showed that compared with the sham group, the cerebral infarction volume was significantly increased while the CBF was reduced remarkably in the MCAO group. 1,25-D3 reduced cerebral infarction volume, increased the recovery of CBF and expressions of VDR, TGF-β, p-Smad2, p-Smad3, and VEGF, significantly increased IB4+ tip cells and CD31+ vascular length in the peri-infarct area compared with the DMSO group. The VDR antagonist pyridoxal-5-phosphate (P5P) partially reversed the neuroprotective effects of 1,25-D3 described above. In summary, 1,25-D3 plays a neuroprotective role in stroke by activating VDR and promoting the activation of TGF-β, which in turn up-regulates the TGF-β/Smad2/3 signaling pathway, increases the release of VEGF and thus promotes angiogenesis, suggesting that this signaling pathway may be an effective target for ischemic stroke treatment. 1,25-D3 is considered to be a neuroprotective agent and is expected to be an effective drug for the treatment of ischemic stroke and related diseases.

Microglia Modulate Cortical Spreading Depolarizations After Ischemic Stroke: A Narrative Review

Cortical spreading depolarizations (CSDs) are characterized by waves of diminished electroencephalography activity that propagate across the cortex with subsequent loss of ionic homeostasis. CSDs have been found in many pathological conditions, including migraine, traumatic brain injury, and ischemic stroke. Because of CSD-associated ionic and metabolic disturbances at the peri-infarct area after ischemic stroke, it is thought that CSDs exacerbate tissue infarction and worsen clinical outcomes. Microglia, the main innate immune cells in the brain, are among the first responders to brain tissue damage. Recent studies demonstrated that microglia play a critical role in CSD initiation and propagation. In this article, we discuss the significance of CSD in the setting of ischemic stroke and how microglia may modulate peri-infarct CSDs, also known as iso-electric depolarizations. Finally, we discuss the significance of microglial Ca2+ and how it might be used as a potential therapeutic target for patients with ischemic stroke.

Astrocyte-secreted chordin-like 1 regulates spine density after ischemic injury

Ischemic injury occurs when the brain is deprived of blood flow, preventing cells from receiving essential nutrients. The injury core is the brain region directly deprived and is surrounded by the peri-infarct area, the region with recovery potential. In the peri-infarct area neurons undergo acute loss of dendritic spines, which modifies synaptic plasticity and determines neuronal survival. Astrocytes can be protective or detrimental to the ischemic injury response depending on the specific stage, yet we lack clear understanding of the underlying mechanisms. Chordin-like 1 (Chrdl1) is an astrocyte-secreted protein that promotes synaptic maturation and limits experience-dependent plasticity in the mouse visual cortex. Given this plasticity-limiting function we asked if Chrdl1 regulates the response to ischemic injury, modelled using photothrombosis (PT). We find that Chrdl1 mRNA is upregulated in astrocytes in the peri-infarct area in both acute and sub-acute phases post-PT. To determine the impact of increased Chrdl1 on the response to PT we analyzed Chrdl1 knock-out mice. We find that absence of Chrdl1 prevents ischemia-induced spine loss in the peri-infarct area and reduces cell death in the core, without impacting gliosis. These findings highlight the important role of astrocyte-secreted proteins in regulating structural plasticity in response to brain ischemic injuries.

Cross-talk between GABAergic postsynapse and microglia regulate synapse loss after brain ischemia.

Microglia interact with neurons to facilitate synapse plasticity; however, signal(s) contributing to microglia activation for synapse elimination in pathology are not fully understood. Here, using in vitro organotypic hippocampal slice cultures and transient middle cerebral artery occlusion (MCAO) in genetically engineered mice in vivo, we report that at 24 hours after ischemia, microglia release brain-derived neurotrophic factor (BDNF) to downregulate glutamatergic and GABAergic synapses within the peri-infarct area. Analysis of the cornu ammonis 1 (CA1) in vitro shows that proBDNF and mBDNF downregulate glutamatergic dendritic spines and gephyrin scaffold stability through p75 neurotrophin receptor (p75NTR) and tropomyosin receptor kinase B (TrkB) receptors, respectively. After MCAO, we report that in the peri-infarct area and in the corresponding contralateral hemisphere, similar neuroplasticity occurs through microglia activation and gephyrin phosphorylation at serine-268 and serine-270 in vivo. Targeted deletion of the Bdnf gene in microglia or GphnS268A/S270A (phospho-null) point mutations protects against ischemic brain damage, neuroinflammation, and synapse downregulation after MCAO.

Knockout of the P2Y6 Receptor Prevents Peri-Infarct Neuronal Loss after Transient, Focal Ischemia in Mouse Brain.

After stroke, there is a delayed neuronal loss in brain areas surrounding the infarct, which may in part be mediated by microglial phagocytosis of stressed neurons. Microglial phagocytosis of stressed or damaged neurons can be mediated by UDP released from stressed neurons activating the P2Y6 receptor on microglia, inducing microglial phagocytosis of such neurons. We show evidence here from a small trial that the knockout of the P2Y6 receptor, required for microglial phagocytosis of neurons, prevents the delayed neuronal loss after transient, focal brain ischemia induced by endothelin-1 injection in mice. Wild-type mice had neuronal loss and neuronal nuclear material within microglia in peri-infarct areas. P2Y6 receptor knockout mice had no significant neuronal loss in peri-infarct brain areas seven days after brain ischemia. Thus, delayed neuronal loss after stroke may in part be mediated by microglial phagocytosis of stressed neurons, and the P2Y6 receptor is a potential treatment target to prevent peri-infarct neuronal loss.