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Background and Purpose: Although VEGF 165 (vascular endothelial growth factor-165) is able to enhance both angiogenesis and neurogenesis, it also increases vascular permeability through the blood-brain barrier. Heparan sulfate (HS) sugars play important roles in regulating VEGF bioactivity in the pericellular compartment. Here we asked whether an affinity-purified VEGF 165 -binding HS (HS7) could augment endogenous VEGF activity during stroke recovery without affecting blood-brain barrier function. Methods: Both rat brain endothelial cell line 4 and primary rat neural progenitor cells were used to evaluate the potential angiogenic and neurogenic effects of HS7 in vitro. For in vivo experiments, male Sprague-Dawley rats were subjected to 100 minutes of transient focal cerebral ischemia, then treated after 4 days with either PBS or HS7. One week later, infarct volume, behavioral sequelae, immunohistochemical markers of angiogenesis and neural stem cell proliferation were assessed. Results: HS7 significantly enhanced VEGF 165 -mediated angiogenesis in rat brain endothelial cell line 4 brain endothelial cells, and increased the proliferation and differentiation of primary neural progenitor cells, both via the VEGFR2 (vascular endothelial growth factor receptor 2) pathway. Intracerebroventricular injection of HS7 improved neurological outcome in ischemic rats without changing infarct volumes. Immunostaining of the compromised cerebrum demonstrated increases in collagen IV/Ki67 and nestin/Ki67 after HS7 exposure, consistent with its ability to promote angiogenesis and neurogenesis, without compromising blood-brain barrier integrity. Conclusions: A VEGF-activating glycosaminoglycan sugar, by itself, is able to enhance endogenous VEGF 165 activity during the post-ischemic recovery phase of stroke.

After stroke, peripheral immune cells are activated and these systemic responses may amplify brain damage, but how the injured brain sends out signals to trigger systemic inflammation remains unclear. Here we show that a brain-to-cervical lymph node (CLN) pathway is involved. In rats subjected to focal cerebral ischemia, lymphatic endothelial cells proliferate and macrophages are rapidly activated in CLNs within 24 h, in part via VEGF-C/VEGFR3 signalling. Microarray analyses of isolated lymphatic endothelium from CLNs of ischemic mice confirm the activation of transmembrane tyrosine kinase pathways. Blockade of VEGFR3 reduces lymphatic endothelial activation, decreases pro-inflammatory macrophages, and reduces brain infarction. In vitro, VEGF-C/VEGFR3 signalling in lymphatic endothelial cells enhances inflammatory responses in co-cultured macrophages. Lastly, surgical removal of CLNs in mice significantly reduces infarction after focal cerebral ischemia. These findings suggest that modulating the brain-to-CLN pathway may offer therapeutic opportunities to ameliorate systemic inflammation and brain injury after stroke., Brain damage induces systemic inflammation, but insights and implication of this induction is still unclear. Here the authors show, using rat and mouse focal cerebral ischemia models, that the damaged brain signals via the VEGF-C/VEFGR3 axis to activate inflammatory responses in the draining cervical lymph nodes to induce systemic inflammation.

As the population increases, energy demands continue to rise rapidly. In order to satisfy this increasing energy demand, biogas offers a potential alternative. Biogas is economically viable to be produced through anaerobic digestion (AD) from various biomass feedstocks that are readily available in Malaysia, such as food waste (FW), palm oil mill effluent (POME), garden waste (GW), landfill, sewage sludge (SS) and animal manure. This paper aims to determine the potential feedstocks for biogas production via AD based on their characteristics, methane yield, kinetic studies and economic analysis. POME and FW show the highest methane yield with biogas yields up to 0.50 L/g VS while the lowest is 0.12 L/g VS by landfill leachate. Kinetic study shows that modified Gompertz model fits most of the feedstock with R 2 up to 1 indicating that this model can be used for estimating treatment efficiencies of full-scale reactors and performing scale-up analysis. The economic analysis shows that POME has the shortest payback period (PBP), highest internal rate of return (IRR) and net present value (NPV). However, it has already been well explored, with 93% of biogas plants in Malaysia using POME as feedstock. The FW generation rate in Malaysia is approximately 15,000 tonnes per day, at the same time FW as the second place shows potential to have a PBP of 5.4 years and 13.3% IRR, which is close to the results achieved with POME. This makes FW suitable to be used as the feedstock for biogas production.

Modulator of apoptosis 1 (MOAP-1) is a Bcl-2-associated X Protein (BAX)-associating protein that plays an important role in regulating apoptosis. It is highly enriched in the brain but its function in this organ remains unknown. Studies on BAX−/− mice suggested that disruption of programmed cell death may lead to abnormal emotional states. We thus hypothesize that MOAP-1−/− mice may also display stress-related behavioral differences and perhaps involved in stress responses in the brain and investigated if a depression-like trait exists in MOAP-1−/− mice, and if so, whether it is age related, and how it relates to central serotonergic stress response in the dorsal raphe nucleus. Young MOAP-1−/− mice exhibit depression-like behavior, in the form of increased immobility time when compared to age-matched wild-type mice in the forced swimming test, which is abolished by acute treatment of fluoxetine. This is supported by data from the tail suspension and sucrose preference tests. Repeated forced swimming stress causes an up-regulation of tryptophan hydroxylase 2 (TPH2) and a down-regulation of brain-derived neurotrophic factor (BDNF) in the dorsal raphe nucleus (DRN) in young wild-type (WT) control mice. In contrast, TPH2 up-regulation was not observed in aged WT mice. Interestingly, such a stress response appears absent in both young and aged MOAP-1−/− mice. Aged MOAP-1−/− and WT mice also have similar immobility times on the forced swimming test. These data suggest that MOAP-1 is required in the regulation of stress response in the DRN. Crosstalk between BDNF and 5-HT appears to play an important role in this stress response. Electronic supplementary material The online version of this article (10.1007/s12035-018-1205-7) contains supplementary material, which is available to authorized users.

Ischemic postconditioning is increasingly being investigated as a therapeutic approach for cerebral ischemia. However, the majority of studies are focused on the acute protection of neurons per se. Whether and how postconditioning affects multiple cells in the recovering neurovascular unit remains to be fully elucidated. Here, we asked whether postconditioning may modulate help-me signaling between injured neurons and reactive microglia. Rats were subjected to 100 min of focal cerebral ischemia, then randomized into a control versus postconditioning group. After 3 days of reperfusion, infarct volumes were significantly reduced in animals treated with postconditioning, along with better neurologic outcomes. Immunostaining revealed that ischemic postconditioning increased expression of vascular endothelial growth factor (VEGF) in neurons within peri-infarct regions. Correspondingly, we confirmed that VEGFR2 was expressed on Iba1-positive microglia/macrophages, and confocal microscopy showed that in postconditioned rats, these cells were polarized to a ramified morphology with higher expression of M2-like markers. Treating rats with a VEGF receptor 2 kinase inhibitor negated these effects of postconditioning on microglia/macrophage polarization. In vitro, postconditoning after oxygen-glucose deprivation up-regulated VEGF release in primary neuron cultures, and adding VEGF to microglial cultures partly shifted their M2-like markers. Altogether, our findings support the idea that after postconditioning, injured neurons may release VEGF as a 'help-me' signal that promotes microglia/macrophage polarization into potentially beneficial phenotypes.

Hydrogen sulfide is believed to be a signalling molecule in the central nervous system. It is known to increase rapidly following an ischemic insult in experimental stroke. Is it protective or deleterious? This review surveys the relevant information available in the literature. It appears that there is no definitive answer to this question at present. Current evidence seems to suggest that the presence of H2S in the ischemic brain may either be deleterious or protective depending on its concentration, deleterious when high and protective when low. Therefore, it can be inferred that either an enhancement or a reduction of its concentration may be of potential use in future stroke therapy.

Despite the efforts in developing therapeutics for stroke, recombinant tissue plasminogen activator (rtPA) remains the only FDA approved drug for ischemic stroke. Regenerative medicine targeting endogenous growth factors has drawn much interest in the clinical field as it provides potential restoration for the damaged brain tissue without being limited by a narrow therapeutic window. To date, most of the translational studies using regenerative medicines have encountered problems and failures. In this review, we discuss the effects of some trophic factors which include of erythropoietin (EPO), brain derived neurotrophic factor (BDNF), granulocyte-colony stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF) and heparin binding epidermal growth factor (HB-EGF) in experimental ischemic stroke models and elaborate the lost in translation of the candidate growth factors from bench to bedside. Several new methodologies have been developed to overcome the caveats in translational studies. This review highlights the latest bioengineering approaches including the controlled release and delivery of growth factors by hydrogel-based scaffolds and the enhancement of half-life and selectivity of growth factors by a novel approach facilitated by glycosaminoglycans.

Hydrogen sulfide is believed to be a signalling molecule in the central nervous system. It is known to increase rapidly following an ischemic insult in experimental stroke. Is it protective or deleterious? This review surveys the relevant information available in the literature. It appears that there is no definitive answer to this question at present. Current evidence seems to suggest that the presence of H2S in the ischemic brain may either be deleterious or protective depending on its concentration, deleterious when high and protective when low. Therefore, it can be inferred that either an enhancement or a reduction of its concentration may be of potential use in future stroke therapy.

Leukotriene B4 (LTB4) has been implicated in ischemic stroke pathology. We examined the prognostic significance of LTB4 levels in patients with acute middle cerebral artery (MCA) infarction and their mechanisms in rat stroke models. In ischemic stroke patients with middle cerebral artery infarction, plasma LTB4 levels were found to increase rapidly, roughly doubling within 24 h when compared to initial post-stroke levels. Further analyses indicate that poor functional recovery is associated with early and more sustained increase in LTB4 rather than the peak levels. Results from studies using a rat embolic stroke model showed increased 5-lipoxygenase (5-LOX) expression in the ipsilateral infarcted cortex compared with sham control or respective contralateral regions at 24 h post-stroke with a concomitant increase in LTB4 levels. In addition, neutrophil influx was also observed in the infarcted cortex. Double immunostaining indicated that neutrophils express 5-LOX and leukotriene A4 hydrolase (LTA4H), highlighting the pivotal contributions of neutrophils as a source of LTB4. Importantly, rise in plasma LTB4 levels corresponded with an increase in LTB4 amount in the infarcted cortex, thereby supporting the use of plasma as a surrogate for brain LTB4 levels. Pre-stroke LTB4 loading increased brain infarct volume in tMCAO rats. Conversely, administration of the 5-LOX-activating protein (FLAP) inhibitor BAY-X1005 or B-leukotriene receptor (BLTR) antagonist LY255283 decreased the infarct volume by a similar extent. To conclude, targeted interruption of the LTB4 pathway might be a viable treatment strategy for acute ischemic stroke. Electronic supplementary material The online version of this article (10.1007/s13311-019-00787-4) contains supplementary material, which is available to authorized users.

Introduction Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD). Emphysema progression attributed not only to alveolar structure loss and pulmonary regeneration impairment, but also to excessive inflammatory response, proteolytic and anti-proteolytic activity imbalance, lung epithelial cells apoptosis, and abnormal lung remodeling. To ameliorate lung damage with higher efficiency in lung tissue engineering and cell therapy, pre-differentiating graft cells into more restricted cell types before transplantation could enhance their ability to anatomically and functionally integrate into damaged lung. In this study, we aimed to evaluate the regenerative and repair ability of lung alveolar epithelium in emphysema model by using lung epithelial progenitors which pre-differentiated from amniotic fluid mesenchymal stem cells (AFMSCs). Methods Pre-differentiation of eGFP-expressing AFMSCs to lung epithelial progenitor-like cells (LEPLCs) was established under a modified small airway growth media (mSAGM) for 7-day induction. Pre-differentiated AFMSCs were intratracheally injected into porcine pancreatic elastase (PPE)-induced emphysema mice at day 14, and then inflammatory-, fibrotic-, and emphysema-related indices and pathological changes were assessed at 6 weeks after PPE administration. Results An optimal LEPLCs pre-differentiation condition has been achieved, which resulted in a yield of approximately 20% lung epithelial progenitors-like cells from AFMSCs in a 7-day period. In PPE-induced emphysema mice, transplantation of LEPLCs significantly improved regeneration of lung tissues through integrating into the lung alveolar structure, relieved airway inflammation, increased expression of growth factors such as vascular endothelial growth factor (VEGF), and reduced matrix metalloproteinases and lung remodeling factors when compared with mice injected with AFMSCs. Histopathologic examination observed a significant amelioration in DNA damage in alveolar cells, detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), the mean linear intercept, and the collagen deposition in the LEPLC-transplanted groups. Conclusion Transplantation of predifferentiated AFMSCs through intratracheal injection showed better alveolar regeneration and reverse elastase-induced pulmonary emphysema in PPE-induced pulmonary emphysema mice. Electronic supplementary material The online version of this article (10.1186/s13287-019-1282-1) contains supplementary material, which is available to authorized users.

Biomaterials provide novel platforms to deliver stem cell and growth factor therapies for central nervous system (CNS) repair. The majority of these approaches have focused on the promotion of neural progenitor cells and neurogenesis. However, it is now increasingly recognized that glial responses are critical for recovery in the entire neurovascular unit. In this study, we investigated the cellular effects of epidermal growth factor (EGF) containing hydrogels on primary astrocyte cultures. Both EGF alone and EGF‐hydrogel equally promoted astrocyte proliferation, but EGF‐hydrogels further enhanced astrocyte activation, as evidenced by a significantly elevated Glial fibrillary acidic protein (GFAP) gene expression. Thereafter, conditioned media from astrocytes activated by EGF‐hydrogel protected neurons against injury and promoted synaptic plasticity after oxygen–glucose deprivation. Taken together, these findings suggest that EGF‐hydrogels can shift astrocytes into neuro‐supportive phenotypes. Consistent with this idea, quantitative‐polymerase chain reaction (qPCR) demonstrated that EGF‐hydrogels shifted astrocytes in part by downregulating potentially negative A1‐like genes (Fbln5 and Rt1‐S3) and upregulating potentially beneficial A2‐like genes (Clcf1, Tgm1, and Ptgs2). Further studies are warranted to explore the idea of using biomaterials to modify astrocyte behavior and thus indirectly augment neuroprotection and neuroplasticity in the context of stem cell and growth factor therapies for the CNS. stem cells translational medicine 2019;8:1242&1248, Biomaterials provide novel platforms to deliver stem cell and growth factor therapies for central nervous system repair. Our data suggest that epidermal growth factor‐containing hydrogels can shift astrocytes into potentially beneficial A2‐like phenotypes that may augment neuroprotection and neuroplasticity during the recovery phase after brain injury.

Stem-cell based therapy is an emerging therapeutic approach for ischemic stroke treatment. Bone marrow stromal cells (BMSCs) are in common use as a cell source for stem cell therapy and show promising therapeutic outcomes for stroke treatment. One challenge is to develop a reliable tracking strategy to monitor the fate of BMSCs and assess their therapeutic effects in order to improve the success rate of such treatment. Herein, TPEEP, a fluorogen with aggregation-induced emission characteristics and near-infrared emission are designed and synthesized and further fabricated into organic nanoparticles (NPs). The obtained NPs show high fluorescence quantum yield, low cytotoxicity with good physical and photostability, which display excellent tracking performance of BMSCs in vitro and in vivo. Using a rat photothrombotic ischemia model as an example, the NP-labeled BMSCs are able to migrate to the stroke lesion site to yield bright red fluorescence. Immunofluorescence staining shows that the NP labeling does not affect the normal function of BMSCs, proving their good biocompatibility in vivo. These merits make TPEEP NP a potential cell tracker to evaluate the fate of BMSCs in cell therapy.

Modulator of apoptosis 1 (MOAP-1) is a Bax-associating protein highly enriched in the brain. In this study, we examined the role of MOAP-1 in promoting ischemic injuries following a stroke by investigating the consequences of MOAP-1 overexpression or deficiency in in vitro and in vivo models of ischemic stroke. MOAP-1 overexpressing SH-SY5Y cells showed significantly lower cell viability following oxygen and glucose deprivation (OGD) treatment when compared to control cells. Consistently, MOAP-1−/− primary cortical neurons were observed to be more resistant against OGD treatment than the MOAP-1+/+ primary neurons. In the mouse transient middle cerebral artery occlusion (tMCAO) model, ischemia triggered MOAP-1/Bax association, suggested activation of the MOAP-1-dependent apoptotic cascade. MOAP-1−/− mice were found to exhibit reduced neuronal loss and smaller infarct volume 24 h after tMCAO when compared to MOAP-1+/+ mice. Correspondingly, MOAP-1−/− mice also showed better integrity of neurological functions as demonstrated by their performance in the rotarod test. Therefore, both in vitro and in vivo data presented strongly support the conclusion that MOAP-1 is an important apoptotic modulator in ischemic injury. These results may suggest that a reduction of MOAP-1 function in the brain could be a potential therapeutic approach in the treatment of acute stroke.

The advance of thrombolytic therapy has been hampered by the lack of optimization of the therapy during the hyperacute phase of focal ischemia. Here, we investigate neurovascular dynamics using a custom-designed hybrid electrocorticography (ECoG)-functional photoacoustic microscopy (fPAM) imaging system during the hyperacute phase (first 6 h) of photothrombotic ischemia (PTI) in male Wistar rats following recombinant tissue plasminogen activator (rtPA)-mediated thrombolysis. We reported, for the first time, the changes in neural activity and cerebral hemodynamic responses following rtPA infusion at different time points post PTI. Interestingly, very early administration of rtPA (< 1 h post PTI) resulted in only partial recovery of neurovascular dynamics (specifically , neural activity recovered to 71 ± 3.5% of baseline and hemodynamics to only 52 ± 2.6% of baseline) and late administration of rtPA (> 4 h post PTI) resulted in the deterioration of neurovascular function. A therapeutic window between 1 and 3 h post PTI was found to improve recovery of neurovascular function (i.e. significant restoration of neural activity to 93 ± 4.2% of baseline and hemodynamics to 81 ± 2.1% of baseline, respectively). The novel combination of fPAM and ECoG enables direct mapping of neurovascular dynamics and serves as a platform to evaluate potential interventions for stroke.

Brain ischemia is a neurological deficit caused by a reduction in the blood supply to tissue, and one of the leading causes of disability in the world. Currently, the most well-known therapeutic agent for ischemia recovery is recombinant tissue plasminogen activator (rtPA), but it is viable for only a small portion (approximately 3.6%) of ischemic patients and may cause side effects such as tissue damage. Thus, introducing a new therapeutic concept for ischemia, we proposed an integrated intervention combining global and focal stimulations in this article. To investigate the potential therapeutic effect of cathodal-transcranial direct current stimulation (C-tDCS) with peripheral sensory stimulation (PSS) during the hyperacute phase of stroke, the present study evaluated neurovascular and neuroprotective responses of the rat cortex following ischemic insult. A hybrid, dual-modality system, including electrocorticography (ECoG) and functional photoacoustic microscopy (fPAM), termed ECoG-fPAM, was used to image cortical functional responses pre- and post-ischemia. Using ECoG-fPAM, results showed that cerebral blood volume (CBV) was able to be recovered during the intervention. In addition, neural activity including somatosensory evoked potentials (SSEPs) and alpha-to-delta ratio (ADR) were restored and greater than the baseline value when the integrated intervention was administered. The results of NeuN/ED-1 immunohistochemical staining and TTC staining also supported the neuroprotective effect of this intervention, protecting more neurons and decreasing the infarct size. Overall, the results acquired from the ECoG-fPAM system demonstrated that C-tDCS + PSS administered immediately following ischemia induction can significantly promote neuroprotection via inhibition of ischemia expansion and reversed cortical neurovascular functions, suggesting effective recovery.

Hydrogen sulfide (H2S) has been reported to exacerbate stroke outcome in experimental models. Cystathionine β-synthase (CBS) has been implicated as the predominant H2S-producing enzyme in central nervous system. When SH-SY5Y cells were transfected to overexpress CBS, these cells were able to synthesize H2S when exposed to high levels of enzyme substrates but not substrate concentrations that may reflect normal physiological conditions. At the same time, these cells demonstrated exacerbated cell death when subjected to oxygen and glucose deprivation (OGD) together with high substrate concentrations, indicating that H2S production has a detrimental effect on cell survival. This effect could be abolished by CBS inhibition. The same effect was observed with primary astrocytes exposed to OGD and high substrates or sodium hydrosulfide. In addition, CBS was upregulated and activated by truncation in primary astrocytes subjected to OGD. When rats were subjected to permanent middle cerebral artery occlusion, CBS activation was also observed. These results imply that in acute ischemic conditions, CBS is upregulated and activated by truncation causing an increased production of H2S, which exacerbate the ischemic injuries. Therefore, CBS inhibition may be a viable approach to stroke treatment.

Organic nanoparticles (NPs) with aggregation-induced emission (AIE) have been successfully used for tracking bone marrow stromal cells (BMSCs) in rats with ischemic stroke, highlighting the great potential of such fluorescent NPs in understanding the fate of transplanted stem cells for cell-based therapies.

Andrographolide is a bioactive molecule isolated from Andrographis paniculata with anticancer and anti-inflammatory activities. In this study, we tested the effects of andrographolide on astrocyte-mediated neuroinflammatory responses. Cultured rat primary astrocytes were treated with proinflammatory cytokine interleukin 1β with or without pretreatment with andrographolide, and then processed for measurements of chemokine C-C motif ligand 5 (CCL5) and glial fibrillary acidic protein. The activation status of nuclear factor-κB activation that may underlie CCL5 upregulation was also measured. Andrographolide pretreatment was found to attenuate the upregulation of CCL5 and glial fibrillary basic protein as well as reduce the phosphorylation of nuclear factor-κB p65 and IκBα after interleukin 1β stimulation. These data suggest that andrographolide should be evaluated further as a therapeutic for central nervous system diseases characterized by astrocyte-mediated neuroinflammatory processes.

3-Mercaptopyruvate sulfurtransferase (3MST) is an important enzyme for the synthesis of hydrogen sulfide (H2S) in the brain. We present here data that indicate an exclusively localization of 3MST in astrocytes. Regional distribution of 3MST activities is even and unremarkable. Following permanent middle cerebral artery occlusion (pMCAO), 3MST was down-regulated in both the cortex and striatum, but not in the corpus collosum. It appears that the down-regulation of astrocytic 3MST persisted in the presence of astrocytic proliferation due to gliosis. Our observations indicate that 3MST is probably not responsible for the increased production of H2S following pMCAO. Therefore, cystathionine β-synthase (CBS), the alternative H2S producing enzyme in the CNS, remains as a more likely potential therapeutic target than 3MST in the treatment of acute stroke through inhibition of H2S production.