Peri-infarct Tissue Research Articles

Mapping the acute time course of immune cell infiltration into an ECM hydrogel in a rat model of stroke using 19F MRI

Extracellular matrix (ECM) hydrogel implantation into a stroke-induced tissue cavity invokes a robust cellular immune response. However, the spatio-temporal dynamics of immune cell infiltration into peri-infarct brain tissues versus the ECM-bioscaffold remain poorly understood. We here tagged peripheral immune cells using perfluorocarbon (PFC) nanoemulsions that afford their visualization by 19F magnetic resonance imaging (MRI). Prior to ECM hydrogel implantation, only blood vessels could be detected using 19F MRI. Using “time-lapse” 19F MRI, we established the infiltration of immune cells into the peri-infarct area occurs 5–6 h post-ECM implantation. Immune cells also infiltrated through the stump of the MCA, as well as a hydrogel bridge that formed between the tissue cavity and the burr hole in the skull. Tissue-based migration into the bioscaffold was observed between 9 and 12 h with a peak signal measured between 12 and 18 h post-implantation. Fluorescence-activated cell sorting of circulating immune cells revealed that 9% of cells were labeled with PFC nanoemulsions, of which the vast majority were neutrophils (40%) or monocytes (48%). Histology at 24 h post-implantation, in contrast, indicated that macrophages (35%) were more numerous in the peri-infarct area than neutrophils (11%), whereas the vast majority of immune cells within the ECM hydrogel were neutrophils (66%). Only a small fraction (12%) of immune cells did not contain PFC nanoemulsions, indicating a low type II error for 19F MRI. 19F MRI hence provides a unique tool to improve our understanding of the spatio-temporal dynamics of immune cells invading bioscaffolds and effecting biodegradation.

Preconditioning With Intermittent Hypobaric Hypoxia Attenuates Stroke Damage and Modulates Endocytosis in Residual Neurons.

Background: Moderate hypobaric hypoxia induces cerebral ischemic tolerance. We investigated the optimal method for applying hypobaric hypoxia preconditioning at 5,000 m to ischemic brain tissue and combined it with proteomics to determine the mechanisms underlying this effect.Methods: Male SD rats were randomly grouped as S (sham, n = 20), M (middle cerebral artery occlusion [MCAO], n = 28), H2M (intermittent hypobaric hypoxia preconditioned MCAO group, 2 h/day, 10 days, n = 20), H6M (intermittent hypobaric hypoxia preconditioned MCAO group, 6 h/day, 10 days, n = 28), and HpM (persistent hypobaric hypoxia preconditioned MCAO group, 10 days, n = 28). The permanent MCAO model was established based on the Zea Longa method. Infarction was assessed with the modified neurological severity score (mNSS) and 2,3,5-triphenyl tetrazolium chloride staining. The total protein expression of the neuron-specific nuclear protein (NeuN), cysteinyl aspartate specific proteinase 3 (caspase-3), cleaved-caspase-3, and interleukin 6 (IL-6) was determined using western blotting. We assessed the peri-infarct cortex's ultrastructural changes. A label-free proteomic study and western blot verification were performed on the most effective preconditioned group.Results: The H6M group showed a lower infarct volume (p = 0.0005), lower mNSS score (p = 0.0009) than the M group. The H2M showed a lower level of IL-6 (p = 0.0213) than the M group. The caspase-3 level decreased in the H2M (p = 0.0002), H6M (p = 0.0025), and HpM groups (p = 0.0054) compared with that in the M group. Cleaved-caspase-3 expression decreased in the H2M (p = 0.0011), H6M (p < 0.0001), and HpM groups (p < 0.0001) compared with that in the M group. The neurons' ultrastructure and the blood-brain barrier in the peri-infarct tissue improved in the H2M and H6M groups. Immunofluorescence revealed increased NeuN-positive cells in the peri-infarct tissue in the H6M group (p = 0.0003, H6M vs. M). Protein expression of Chmp1a, Arpc5, and Hspa2 factors related to endocytosis were upregulated in the H6M compared with those of the M group (p < 0.05 for all) on western blot verification of label-free proteomics.Conclusions: Intermittent hypobaric hypoxia preconditioning exerts a neuroprotective effect in a rat stroke model. Persistent hypobaric hypoxia stimulation exhibited no significant neuroprotective effect. Intermittent hypoxic preconditioning for 6 h/day for 10 days upregulates key proteins in clathrin-dependent endocytosis of neurons in the cortex.

CHIP ameliorates cerebral ischemia-reperfusion injury by attenuating necroptosis and inflammation.

Blood reperfusion of ischemic cerebral tissue may cause cerebral ischemia-reperfusion (CIR) injury. Necroptosis and inflammation have been demonstrated to be involved in the disease-related process of CIR injury. The E3 ubiquitin ligase carboxyl terminus of Hsp70-interacting protein (CHIP) can modulate multiple cellular signaling processes, including necroptosis and inflammation. Numerous studies have demonstrated the neuroprotective effects of CHIP on multiple central nervous system (CNS) diseases. However, the effects of CHIP on CIR injury have not been fully explored. We hypothesize that CHIP can exert neuroprotective effects by attenuating necroptosis and inflammation during CIR injury. In the present study, adult wild-type (WT) C57BL/6 mice and CHIP knock-in (KI) mice with a C57BL/6 background and CHIP overexpression in neural tissue underwent middle cerebral artery occlusion (MCAO) surgery to simulate CIR onset. Our data indicated that CHIP expression in the peri-infarct tissue was markedly increased after MCAO surgery. Compared with WT mice, CHIP KI mice significantly improved neurological deficit scores, decreased cerebral infarct volume, and attenuated brain edema and neuronal damage. Meanwhile, CHIP overexpression attenuated necroptosis and inflammation induced by MCAO surgery. These findings indicated that overexpression of CHIP might exert neuroprotective effects by attenuating necroptosis and inflammation during CIR injury, and increasing CHIP levels may be a potential strategy in cerebrovascular disease therapy.

Physical therapy exerts sub-additive and suppressive effects on intracerebral neural stem cell implantation in a rat model of stroke

Intracerebral cell therapy (CT) is emerging as a new therapeutic paradigm for stroke. However, the impact of physical therapy (PT) on implanted cells and their ability to promote recovery remains poorly understood. To address this translational issue, a clinical-grade neural stem cell (NSC) line was implanted into peri-infarct tissue using MRI-defined injection sites, two weeks after stroke. PT in the form of aerobic exercise (AE) was administered 5 × per week post-implantation using a paradigm commonly applied in patients with stroke. A combined AE and CT exerted sub-additive therapeutic effects on sensory neglect, whereas AE suppressed CT effects on motor integration and grip strength. Behavioral testing emerged as a potentially major component for task integration. It is expected that this study will guide and inform the incorporation of PT in the design of clinical trials evaluating intraparenchymal NSCs implantation for stroke.

Mesenchymal stromal cell-derived small extracellular vesicles promote neurological recovery and brain remodeling after distal middle cerebral artery occlusion in aged rats

Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) promote neurological recovery after middle cerebral artery occlusion (MCAO) in young rodents. Ischemic stroke mainly affects aged humans. MSC-sEV effects on stroke recovery in aged rodents had not been assessed. In a head-to-head comparison, we exposed young (4–5 months) and aged (19–20 months) male Sprague–Dawley rats to permanent distal MCAO. At 24 h, 3 and 7 days post-stroke, vehicle or MSC-sEVs (2 × 106 or 2 × 107 MSC equivalents/kg) were intravenously administered. Neurological deficits, ischemic injury, brain inflammatory responses, post-ischemic angiogenesis, and endogenous neurogenesis were evaluated over 28 days. Post-MCAO, aged vehicle-treated rats exhibited more severe motor-coordination deficits evaluated by rotating pole and cylinder tests and larger brain infarcts than young vehicle-treated rats. Although infarct volume was not influenced by MSC-sEVs, sEVs at both doses effectively reduced motor-coordination deficits in young and aged rats. Brain macrophage infiltrates in periinfarct tissue, which were evaluated as marker of a recovery-aversive inflammatory environment, were significantly stronger in aged than young vehicle-treated rats. sEVs reduced brain macrophage infiltrates in aged, but not young rats. The tolerogenic shift in immune balance paved the way for structural brain tissue remodeling. Hence, sEVs at both doses increased periinfarct angiogenesis evaluated by CD31/BrdU immunohistochemistry in young and aged rats, and low-dose sEVs increased neurogenesis in the subventricular zone examined by DCX/BrdU immunohistochemistry. Our study provides robust evidence that MSC-sEVs promote functional neurological recovery and brain tissue remodeling in aged rats post-stroke. This study encourages further proof-of-concept studies in clinic-relevant stroke settings.

Distinct metabolic transcriptional patterns and Warburg effect in porcine model of ischaemic cardiomyopathy

Abstract Background Despite improvements in therapy for heart failure, the prognosis remains poor. Energetic imbalances related to mitochondrial dysfunction, impaired oxidative phosphorylation and oxidative damage have been implicated in the pathogenesis or worsening of heart failure. Improved understanding of the metabolic alterations of the heart may provide new biomarkers or therapeutic targets. Purpose It has been speculated that aerobic glycolysis, known as the Warburg effect, may be advantageous for cell survival whereby there is the opportunity for simultaneous energy production and production of intermediary metabolites crucial for anabolic pathways that sustain cell division and maintenance, and protein synthesis (1,2). In this study, we studied a preclinical model of ischaemic cardiomyopathy to investigate transcriptional patterns in two defined areas; peri-infarct and remote locations. Specifically, we investigated if aerobic glycolysis may be present in this setting. Methods 18 female Yorkshire pigs were studied. 10 animals underwent balloon catheter myocardial infarction followed by termination at 4 weeks. A group of 8 healthy animals served as controls. All animals underwent cardiac MRI prior to termination confirming ventricular impairment and remodelling in the chronic animals (see Table 1). Gene expression profiles of glucose mobilisation and glucose metabolic markers (GLUT1, GLUT4, ANK2, GAPDH and LDHA), oxidative phosphorylation and mitochondrial function (ND1, TFAM, and PGC1-alpha), key markers of insulin resistance (AKT1, AS160) and a hypoxia marker (HIF1-alpha) were measured. Results Our results indicate the presence of two distinct metabolic profiles at the gene expression level, differing from control tissue but more interestingly with striking differences regionally. In the remote region compared to controls, we demonstrate a picture suggestive of increased glycolysis with significant transcriptional upregulation of GLUT1 and GLUT4 glucose transporters (p=0.01 and p=0.02 respectively), alongside a significant reduction in PGC1-alpha (p=0.04) with maintained transcriptional levels of LDHA and HIF1-alpha. Combined, these results are suggestive of reduced oxidative phosphorylation alongside compensated, or early stage, aerobic glycolytic metabolism in this region. In contrast, in peri-infarct tissue versus controls, we demonstrate a reduction in glycolysis (GLUT1 p=0.09, GLUT4 p=0.01 with a dramatic increase in GLUT1/GLUT4 ratio of 6.7 times) and reduced markers of mitochondrial function indicative of insulin-resistance with evidence of a fetal-pattern metabolic profile (see Table 2 for detailed results). Conclusions This study demonstrates distinct transcriptional profiles within hearts with ischaemic cardiomyopathy and for the first time suggests that the Warburg effect may be present in remote myocardium in this setting supporting the need for further work in this area. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): This research work was supported by grants awarded to Professor Ascione: the British Heart Foundation (BHF) (BHF IG/14/2/30991, BHF RM/13/2/30158), and the Medical Research Council (MRC) (MRC MR/L012723/1). Table 1. CMR parameters for animals studiedTable 2. Transcriptional signals

Post-acute delivery of α5-GABAA antagonist, S 44819, improves functional recovery in juvenile rats following stroke

Hypo-excitability was reported in the peri-infarct tissue following stroke, an effect counteracted by a blockage of α5-GABAA receptors in adult rodents. Our present study aims to evaluate the effect of a selective α5-GABAA receptor antagonist, S 44819, in stroke in juvenile animals. We have set up and characterized an original model of transient ischemic stroke in 28 day-old Sprague-Dawley rats (45-min occlusion of the middle cerebral artery by intraluminal suture). In this model, S 44819 (1, 3 and 10 mg/kg, b.i.d) was orally administered from day 3 to day 16 after stroke onset. Sensorimotor recovery was assessed on day 1, day 9 and day 16 after stroke onset. Results show that rats treated with S 44819 at the doses of 3 and 10 mg/kg displayed a significant improvement of the neurological deficits (neuroscore) on day 9 and day 16, when compared with animals treated with vehicle. Grip-test data analysis reveals that rats treated with S 44819 at the dose of 3 mg/kg displayed a better recovery on day 9 and day 16. These results are in agreement with those previously observed in adult rats, demonstrating that targeting α5-GABAA receptors improves neurological recovery after stroke in juvenile rats.

Effect of Anti-inflammatory Treatment with AMD3100 and CX3CR1 Deficiency on GABAA Receptor Subunit and Expression of Glutamate Decarboxylase Isoforms After Stroke

Following stroke, attenuation of detrimental inflammatory pathways might be a promising strategy to improve long-term outcome. In particular, cascades driven by pro-inflammatory chemokines interact with neurotransmitter systems such as the GABAergic system. This crosstalk might be of relevance for mechanisms of neuronal plasticity, however, detailed studies are lacking. The purpose of this study was to determine if treatment with 1,1′-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (AMD3100), an antagonist to the C-X-C chemokine receptor type 4 (CXCR4) and partial allosteric agonist to CXCR7 (AMD3100) alone or in combination with C-X3-C chemokine receptor type 1 (CX3CR1) deficiency, affect the expression of GABAA subunits and glutamate decarboxylase (GAD) isoforms. Heterozygous, CX3CR1-deficient mice and wild-type littermates were subjected to photothrombosis (PT). Treatment with AMD3100 (0.5 mg/kg twice daily i.p.) was administered starting from day 2 after induction of PT until day 14 after the insult. At this time point, GABAA receptor subunits (α3, β3, δ), GAD65 and GAD67, and CXCR4 were analyzed from the peri-infarct tissue and homotypic brain regions of the contralateral hemisphere by quantitative real-time PCR and Western Blot. Fourteen days after PT, CX3CR1 deficiency resulted in a significant decrease of the three GABAA receptor subunits in both the lesioned and the contralateral hemisphere compared to sham-operated mice. Treatment with AMD3100 promoted the down-regulation of GABAA subunits and GAD67 in the ipsilateral peri-infarct area, while the β3 subunit and the GAD isoforms were up-regulated in homotypic regions of the contralateral cortex. Changes in GABAA receptor subunits and GABA synthesis suggest that the CXCR4/7 and CX3CR1 signaling pathways are involved in the regulation of GABAergic neurotransmission in the post-ischemic brain.

Quantitative susceptibility mapping in ischemic stroke patients after successful recanalization

Quantitative susceptibility mapping (QSM) is a novel processing method for gradient-echo magnetic resonance imaging (MRI). Higher magnetic susceptibility in cortical veins have been observed on susceptibility maps in the ischemic hemisphere of stroke patients, indicating an increased oxygen extraction fraction (OEF). Our goal was to investigate susceptibility in veins of stroke patients after successful recanalization in order to analyze the value of QSM in predicting tissue prognosis and clinical outcome. We analyzed MR images of 23 patients with stroke due to unilateral middle cerebral artery (MCA)-M1/M2 occlusion acquired 24–72 h after successful thrombectomy. The susceptibilities of veins were obtained from QSM and compared between the stroke territory, the ipsilateral non-ischemic MCA territory and the contralateral MCA territory. As outcome variables, early infarct size and functional disability (modified Rankin Scale, mRS) after 3–5 months was used. The median susceptibility value of cortical veins in the ischemic core was 41% lower compared to the ipsilateral non-ischemic MCA territory and 38% lower than on the contralateral MCA territory. Strikingly, in none of the patients prominent vessels with high susceptibility signal were found after recanalization. Venous susceptibility values within the infarct did not correlate with infarct volume or functional disability after 3–5 months. Low venous susceptibility within the infarct core after successful recanalization of the occluded vessel likely indicates poor oxygen extraction arising from tissue damage. We did not identify peri-infarct tissue with increased susceptibility values as potential surrogate of former penumbral areas. We found no correlation of QSM parameters with infarct size or outcome.

Differential effects of the cell cycle inhibitor, olomoucine, on functional recovery and on responses of peri-infarct microglia and astrocytes following photothrombotic stroke in rats

BackgroundFollowing stroke, changes in neuronal connectivity in tissue surrounding the infarct play an important role in both spontaneous recovery of neurological function and in treatment-induced improvements in function. Microglia and astrocytes influence this process through direct interactions with the neurons and as major determinants of the local tissue environment. Subpopulations of peri-infarct glia proliferate early after stroke providing a possible target to modify recovery. Treatment with cell cycle inhibitors can reduce infarct volume and improve functional recovery. However, it is not known whether these inhibitors can influence neurological function or alter the responses of peri-infarct glia without reducing infarction. The present study aimed to address these issues by testing the effects of the cell cycle inhibitor, olomoucine, on recovery and peri-infarct changes following photothrombotic stroke.MethodsStroke was induced by photothrombosis in the forelimb sensorimotor cortex in Sprague-Dawley rats. Olomoucine was administered at 1 h and 24 h after stroke induction. Forelimb function was monitored up to 29 days. The effects of olomoucine on glial cell responses in peri-infarct tissue were evaluated using immunohistochemistry and Western blotting.ResultsOlomoucine treatment did not significantly affect maximal infarct volume. Recovery of the affected forelimb on a placing test was impaired in olomoucine-treated rats, whereas recovery in a skilled reaching test was substantially improved. Olomoucine treatment produced small changes in aspects of Iba1 immunolabelling and in the number of CD68-positive cells in cerebral cortex but did not selectively modify responses in peri-infarct tissue. The content of the astrocytic protein, vimentin, was reduced by 30% in the region of the lesion in olomoucine-treated rats.ConclusionsOlomoucine treatment modified functional recovery in the absence of significant changes in infarct volume. The effects on recovery were markedly test dependent, adding to evidence that skilled tasks requiring specific training and general measures of motor function can be differentially modified by some interventions. The altered recovery was not associated with specific changes in key responses of peri-infarct microglia, even though these cells were considered a likely target for early olomoucine treatment. Changes detected in peri-infarct reactive astrogliosis could contribute to the altered patterns of functional recovery.

Ki20227 aggravates apoptosis, inflammatory response, and oxidative stress after focal cerebral ischemia injury.

The survival of microglia depends on the colony-stimulating factor-1 receptor (CSF1R) signaling pathway under physiological conditions. Ki20227 is a highly selective CSF1R inhibitor that has been shown to change the morphology of microglia. However, the effects of Ki20227 on the progression of ischemic stroke are unclear. In this study, male C57BL/6 mouse models of focal cerebral ischemic injury were established through the occlusion of the middle cerebral artery and then administered 3 mg/g Ki20227 for 3 successive days. The results revealed that the number of ionized calcium-binding adaptor molecule 1/bromodeoxyuridine double positive cells in the infarct tissue was reduced, the degree of edema was increased, neurological deficits were aggravated, infarct volume was increased, and the number of peri-infarct Nissl bodies was reduced. The number of terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells in the peri-infarct tissue was increased. The expression levels of Bax and Cleaved caspase-3 were up-regulated. Bcl-2 expression was downregulated. The expression levels of inflammatory factors and oxidative stress-associated factors were increased. These findings suggested that Ki20227 blocked microglial proliferation and aggravated the pathological progression of ischemia/reperfusion injury in a transient middle cerebral artery occlusion model. This study was approved by the Animal Ethics Committee of Lanzhou University Second Hospital (approval No. D2020-68) on March 6, 2020.

Low-Dose Empagliflozin Improves Systolic Heart Function after Myocardial Infarction in Rats: Regulation of MMP9, NHE1, and SERCA2a.

The effects of the selective sodium-glucose cotransporter 2 (SGLT2) inhibitor empagliflozin in low dose on cardiac function were investigated in normoglycemic rats. Cardiac parameters were measured by intracardiac catheterization 30 min after intravenous application of empagliflozin to healthy animals. Empagliflozin increased the ventricular systolic pressure, mean pressure, and the max dP/dt (p < 0.05). Similarly, treatment with empagliflozin (1 mg/kg, p.o.) for one week increased the cardiac output, stroke volume, and fractional shortening (p < 0.05). Myocardial infarction (MI) was induced by ligation of the left coronary artery. On day 7 post MI, empagliflozin (1 mg/kg, p.o.) improved the systolic heart function as shown by the global longitudinal strain (−21.0 ± 1.1% vs. −16.6 ± 0.7% in vehicle; p < 0.05). In peri-infarct tissues, empagliflozin decreased the protein expression of matrix metalloproteinase 9 (MMP9) and favorably regulated the cardiac transporters sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) and sodium hydrogen exchanger 1 (NHE1). In H9c2 cardiac cells, empagliflozin decreased the MMP2,9 activity and prevented apoptosis. Empagliflozin did not alter the arterial stiffness, blood pressure, markers of fibrosis, and necroptosis. Altogether, short-term treatment with low-dose empagliflozin increased the cardiac contractility in normoglycemic rats and improved the systolic heart function in the early phase after MI. These effects are attributed to a down-regulation of MMP9 and NHE1, and an up-regulation of SERCA2a. This study is of clinical importance because it suggests that a low-dose treatment option with empagliflozin may improve cardiovascular outcomes post-MI. Down-regulation of MMPs could be relevant to many remodeling processes including cancer disease.

Neuroprotective and antioxidative effects of pioglitazone in brain tissue adjacent to the ischemic core are mediated by PI3K/Akt and Nrf2/ARE pathways

The present study elucidates the neuroprotective mechanisms of the PPARγ (peroxisome proliferator-activated receptor γ) agonist pioglitazone in survival of ischemic neurons following middle cerebral artery occlusion with reperfusion (MCAO). Intracerebroventricular infusion of pioglitazone over 5 days before and 24 or 48 h after MCAO alleviated neurological impairments, inhibited apoptosis 24 h, and activated the PI3K/Akt pathway along with increased phosphorylation of Akt (ser473) and GSK-3β (ser9) in the peri-infarct cortical areas 48 h after MCAO. In primary cortical neurons, pioglitazone suppressed the glutamate-induced release of lactate dehydrogenase by a PPARγ-dependent mechanism. This protective effect was reversed after co-treatment with PI3K and Akt inhibitors, LY294002 and SH-6, respectively. Pioglitazone enhanced the expression of the antioxidative transcription factor Nrf2 and its target gene protein, heme oxidase-1, in the peri-infarct area. Pioglitazone also increased activation of the antioxidant response element (ARE) in neuronal PC12 cells transfected with the pNQO1-rARE plasmid. We demonstrate in primary cortical neurons from Nrf2 knockout mice that the lack of Nrf2 completely abolished the neuroprotective effects of pioglitazone against oxidative and excitotoxic damage. Our results strongly suggest that the neuroprotective effects of PPARγ in peri-infarct brain tissues comprise the concomitant activation of the PI3K/Akt and Nrf2/ARE pathways.Key messagesPioglitazone inhibits apoptosis in ischemic brain tissue. Pioglitazone acting on PPARγ activates PI3K/Akt pathway in ischemic brain tissue.Pioglitazone activates via Nrf2 the antioxidant defense pathway in injured neurons.Pioglitazone activates the antioxidant response element in neuronal PC12 cells.Pioglitazone fails to protect primary neurons lacking Nrf2 against oxidative damage.Activation of PPARγ supports the survival of viable neurons in peri-infarct regions.

Targeting GABAC Receptors Improves Post-Stroke Motor Recovery.

Ischemic stroke remains a leading cause of disability worldwide, with limited treatment options available. This study investigates GABAC receptors as novel pharmacological targets for stroke recovery. The expression of ρ1 and ρ2 mRNA in mice were determined in peri-infarct tissue following photothrombotic motor cortex stroke. (R)-4-amino-cyclopent-1-enyl butylphosphinic acid (R)-4-ACPBPA and (S)-4-ACPBPA were assessed using 2-elecotrode voltage electrophysiology in Xenopus laevis oocytes. Stroke mice were treated for 4 weeks with either vehicle, the α5-selective negative allosteric modulator, L655,708, or the ρ1/2 antagonists, (R)-4-ACPBPA and (S)-4-ACPBPA respectively from 3 days post-stroke. Infarct size and expression levels of GAT3 and reactive astrogliosis were determined using histochemistry and immunohistochemistry respectively, and motor function was assessed using both the grid-walking and cylinder tasks. After stroke, significant increases in ρ1 and ρ2 mRNAs were observed on day 3, with ρ2 showing a further increase on day 7. (R)- and (S)-4-ACPBPA are both potent antagonists at ρ2 and only weak inhibitors of α5β2γ2 receptors. Treatment with either L655,708, (S)-4-ACPBPA (ρ1/2 antagonist; 5 mM only), or (R)-4-ACPBPA (ρ2 antagonist; 2.5 and 5 mM) from 3 days after stroke resulted in a significant improvement in motor recovery on the grid-walking task, with L655,708 and (R)-4-ACPBPA also showing an improvement in the cylinder task. Infarct size was unaffected, and only (R)-4-ACPBPA significantly increased peri-infarct GAT3 expression and decreased the level of reactive astrogliosis. Importantly, inhibiting GABAC receptors affords significant improvement in motor function after stroke. Targeting the ρ-subunit could provide a novel delayed treatment option for stroke recovery.

Abstract P794: Neuregulin-1 Induction of a Distinct Neuronal Growth Program Profile Post-Ischemia

Stroke is ranked as the fifth leading cause of death and the leading cause of adult disability in the United States. Tissue Plasminogen Activator (tPA) is the only FDA approved stroke treatment. Unfortunately, tPA is only administered to 2-5% of patients due to risk of hemorrhage, and it has no therapeutic effect on neuroprotection or neurological recovery. Thus, there is no treatment capable of addressing progressive adult disability. However, it has been shown that exogenous Neuregulin-1 (NRG-1) significantly decreases acute neuronal death and neuroinflammation after ischemic injury. Furthermore, NRG-1 has demonstrated a neuroregenerative effect with increased neuronal sprouting, synapse formation markers, and functional recovery after ischemia. However, the spatiotemporal regenerative effects of NRG-1 following ischemia have not been clearly elucidated. This study aimed to define the endogenous and NRG-1 treatment induced neuronal growth program (NGP) by spatiotemporally characterizing neuroplasticity and neuronal gene expression after ischemic stroke. Male and Female Thy1-eGFP+ transgenic mice were given middle cerebral artery occlusion (MCAO). Mice were treated daily for 10 days, I.P., with vehicle (0.1% BSA, endogenous) or NRG-1 (5ug/kg, treatment-induced) starting 24 hours after MCAO and sacrificed at 10dpi. Confocal microscopy data indicate that NRG-1 daily I.P. treatment protects Thy1-eGFP+ cortical neurons and exhibits more processes and less debris within the peri-infarct tissue. NRG-1 treated mice also exhibited an increase in BCAS1+/Olig2+ oligodendrocytes within the ipsilateral cortex and Olig2+ cells in the ipsilateral subcortex and contralateral cortex compared to vehicle treated mice 24 hours after MCAO. Additional studies including digital spatial profiling (DSP) suggest a spatiotemporal regulation of transcriptional profiles initiated by endogenous and NRG-1 treatment-induced NGP. These studies will help determine whether NRG-1 amplifies endogenous neuroplasticity or induces a distinct and/or region-specific neuroplastic pathway.

Delayed Treatment with Human Dental Pulp Stem Cells Accelerates Functional Recovery and Modifies Responses of Peri-Infarct Astrocytes Following Photothrombotic Stroke in Rats

Dental pulp contains multipotent mesenchymal stem cells that improve outcomes when administered early after temporary middle cerebral artery occlusion in rats. To further assess the therapeutic potential of these cells, we tested whether functional recovery following stroke induced by photothrombosis could be modified by a delayed treatment that was initiated after the infarct attained maximal volume. Photothrombosis induces permanent focal ischemia resulting in tissue changes that better reflect key aspects of the many human strokes in which early restoration of blood flow does not occur. Human dental pulp stem cells (approximately 400 × 103 viable cells) or vehicle were injected into the infarct and adjacent brain tissue of Sprague-Dawley rats at 3 days after the induction of unilateral photothrombotic stroke in the sensorimotor cortex. Forepaw function was tested up to 28 days after stroke. Cellular changes in peri-infarct tissue at 28 days were assessed using immunohistochemistry. Rats treated with the stem cells showed faster recovery compared with vehicle-treated animals in a test of forelimb placing in response to vibrissae stimulation and in first attempt success in a skilled forelimb reaching test. Total success in the skilled reaching test and forepaw use during exploration in a Perspex cylinder were not significantly different between the 2 groups. At 28 days after stroke, rats treated with the stem cells showed decreased immunolabeling for glial fibrillary acidic protein in tissue up to 1 mm from the infarct, suggesting decreased reactive astrogliosis. Synaptophysin, a marker of synapses, and collagen IV, a marker of capillaries, were not significantly altered at this time by the stem-cell treatment. These results indicate that dental pulp stem cells can accelerate recovery without modifying initial infarct formation. Decreases in reactive astrogliosis in peri-infarct tissue could have contributed to the change by promoting adaptive responses in neighboring neurons.

DL-3-n-butylphthalide Increases Collateriogenesis and Functional Recovery after Focal Ischemic Stroke in Mice.

Recent evidence indicates that collateral circulation is critical for the outcome of ischemic stroke. DL-3-n-butylphthalide (NBP), a synthesized compound based on an extract from seeds of celery Apium graveolens Linn, has been used as a therapeutic drug, showing multiple neuroprotective and regenerative activities. A potential effect of NBP on collateral arterial regulation is unknown. We examined the effects of NBP on arteriogenesis of collateral arteries in vitro and a mouse ischemic stroke model. In cultures of mouse iPS cell-derived vascular progenitors, NBP (10 μM) significantly increased α-smooth muscle actin (αSMA)/CD-31 co-labeled cells and the expression of newly formed vasculature marker PDGFRα. A sensorimotor cortex ischemia was induced in transgenic mice expressing αSMA-GFP that allowed direct observation of arterial vasculatures in brain regions. NBP (80 mg/kg) was intranasally delivered 1 hr after stroke and once daily for 14 days. To label proliferating cells, 5-Bromo-2’-deoxyuridine (BrdU, 50 mg/kg, i.p.) was administrated every day from 3 days after stroke. Western blotting of peri-infarct tissue detected increased expressions of VEGF, Ang-1 and reduced nNOS level in NBP-treated mice. The NBP treatment significantly increased αSMA/BrdU co-labeled cells, the diameter of ipsilateral collaterals, and arterial area in ischemic and peri-infarct regions examined 14 days after stroke. Examined 3 days after stroke, NBP prevented functional deficits in the cylinder test and corner test. The NBP treatment of 14 days improved the local cerebral blood flow (LCBF) and functional performance in multiple tests. Thus, NBP promotes collateriogenesis, short and long-term structural and functional improvements after ischemic stroke.

ECM hydrogel improves the delivery of PEG microsphere-encapsulated neural stem cells and endothelial cells into tissue cavities caused by stroke

Intracerebral implantation of neural stem cells (NSCs) to treat stroke remains an inefficient process with <5% of injected cells being retained. To improve the retention and distribution of NSCs after a stroke, we investigated the utility of NSCs’ encapsulation in polyethylene glycol (PEG) microspheres. We first characterized the impact of the physical properties of different syringes and needles, as well as ejection speed, upon delivery of microspheres to the stroke injured rat brain. A 20 G needle size at a 10 μL/min flow rate achieved the most efficient microsphere ejection. Secondly, we optimized the delivery vehicles for in vivo implantation of PEG microspheres. The suspension of microspheres in extracellular matrix (ECM) hydrogel showed superior retention and distribution in a cortical stroke caused by photothrombosis, as well as in a striatal and cortical cavity ensuing middle cerebral artery occlusion (MCAo). Thirdly, NSCs or NSCs + endothelial cells (ECs) encapsulated into biodegradable microspheres were implanted into a large stroke cavity. Cells in microspheres exhibited a high viability, survived freezing and transport. Implantation of 110 cells/microsphere suspended in ECM hydrogel produced a highly efficient delivery that resulted in the widespread distribution of NSCs in the tissue cavity and damaged peri-infarct tissues. Co-delivery of ECs enhanced the in vivo survival and distribution of ∼1.1 million NSCs. The delivery of NSCs and ECs can be dramatically improved using microsphere encapsulation combined with suspension in ECM hydrogel. These biomaterial innovations are essential to advance clinical efforts to improve the treatment of stroke using intracerebral cell therapy.

Epothilones Improve Axonal Growth and Motor Outcomes after Stroke in the Adult Mammalian CNS

SummaryStroke leads to the degeneration of short-range and long-range axonal connections emanating from peri-infarct tissue, but it also induces novel axonal projections. However, this regeneration is hampered by growth-inhibitory properties of peri-infarct tissue and fibrotic scarring. Here, we tested the effects of epothilone B and epothilone D, FDA-approved microtubule-stabilizing drugs that are powerful modulators of axonal growth and scar formation, on neuroplasticity and motor outcomes in a photothrombotic mouse model of cortical stroke. We find that both drugs, when administered systemically 1 and 15 days after stroke, augment novel peri-infarct projections connecting the peri-infarct motor cortex with neighboring areas. Both drugs also increase the magnitude of long-range motor projections into the brainstem and reduce peri-infarct fibrotic scarring. Finally, epothilone treatment induces an improvement in skilled forelimb motor function. Thus, pharmacological microtubule stabilization represents a promising target for therapeutic intervention with a wide time window to ameliorate structural and functional sequelae after stroke.

Hypocaloric Diet Initiated Post-Ischemia Provides Long-Term Neuroprotection and Promotes Peri-Infarct Brain Remodeling by Regulating Metabolic and Survival-Promoting Proteins

Calorie restriction confers post-ischemic neuroprotection, when administered in a defined time window before ischemic stroke. How a hypocaloric diet influences stroke recovery when initiated after stroke has not been investigated. Male C57BL6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. Immediately post-ischemia, mice were randomized to two groups receiving moderately hypocaloric (2286 kcal/kg food) or normocaloric (3518 kcal/kg) diets ad libitum. Animals were sacrificed at 3 or 56 days post-ischemia (dpi). Besides increased low density lipoprotein at 3 days and reduced alanine aminotransferase and increased urea at 56 days, no alterations of plasma markers were found in ischemic mice on hypocaloric diet. Body weight mildly decreased over 56 dpi by 7.4%. Hypocaloric diet reduced infarct volume in the acute stroke phase at 3 dpi and decreased brain atrophy, increased neuronal survival and brain capillary density in peri-infarct striatum and reduced motor coordination impairment in tight rope tests in the post-acute stroke phase over up to 56 dpi. The abundance of brain-derived neurotrophic factor, the NAD-dependent deacetylase and longevity protein sirtuin-1, the anti-oxidant glutathione peroxidase-3, and the ammonium detoxifier glutamine synthetase in the peri-infarct brain tissue was increased by hypocaloric diet. This study shows that a moderately hypocaloric diet that is initiated after stroke confers long-term neuroprotection and promotes peri-infarct brain remodeling.