Adeno-Associated Virus-Mediated Dorsal Root Ganglion Toxicity in the New Zealand White Rabbit.

Recombinant adeno-associated virus (AAV)-mediated degeneration of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (TG) has been observed in non-human primates (NHPs) following intravenous (IV) and intrathecal (IT) delivery. Administration of recombinant AAV encoding a human protein transgene via a single intra-cisterna magna (ICM) injection in New Zealand white rabbits resulted in histopathology changes very similar to NHPs: mononuclear cell infiltration, degeneration/necrosis of sensory neurons, and nerve fiber degeneration of sensory tracts in the spinal cord and of multiple nerves. AAV-associated clinical signs and incidence/severity of histologic findings indicated that rabbits were equally or more sensitive than NHPs to sensory neuron damage. Another study using human and rabbit transgene constructs of the same protein demonstrated comparable changes suggesting that the effects are not an immune response to the non-self protein transgene. Rabbit has not been characterized as a species for general toxicity testing of AAV gene therapies, but these studies suggest that it may be an alternative model to investigate mechanisms of AAV-mediated neurotoxicity and test novel AAV designs mitigating these adverse effects.

Vascular Endothelial Growth Factor 165-Binding Heparan Sulfate Promotes Functional Recovery From Cerebral Ischemia.

BACKGROUND AND PURPOSE: Although VEGF165 (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 VEGF165-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 VEGF165-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 VEGF165 activity during the post-ischemic recovery phase of stroke.

Protective Effects of Endothelial Progenitor Cell-Derived Extracellular Mitochondria in Brain Endothelium.

Endothelial progenitor cells (EPCs) have been pursued as a potential cellular therapy for stroke and central nervous system injury. However, their underlying mechanisms remain to be fully defined. Recent experimental studies suggest that mitochondria may be released and transferred between cells. In this proof-of-concept study, we asked whether beneficial effects of EPCs may partly involve a mitochondrial phenomenon as well. First, EPC-derived conditioned medium was collected and divided into supernatant and particle fractions after centrifugation. Electron microscopy, Western blots, and flow cytometry showed that EPCs were able to release mitochondria. ATP and oxygen consumption assays suggested that these extracellular mitochondria may still be functionally viable. Confocal microscopy confirmed that EPC-derived extracellular mitochondria can be incorporated into normal brain endothelial cells. Adding EPC particles to brain endothelial cells promoted angiogenesis and decreased the permeability of brain endothelial cells. Next, we asked whether EPC-derived mitochondria may be protective. As expected, oxygen-glucose deprivation (OGD) increased brain endothelial permeability. Adding EPC-derived mitochondria particles to the damaged brain endothelium increased levels of mitochondrial protein TOM40, mitochondrial DNA copy number, and intracellular ATP. Along with these indirect markers of mitochondrial transfer, endothelial tightness was also restored after OGD. Taken together, these findings suggest that EPCs may support brain endothelial energetics, barrier integrity, and angiogenic function partly through extracellular mitochondrial transfer. Stem Cells 2018;36:1404-1410.

Effects of ischemic post-conditioning on neuronal VEGF regulation and microglial polarization in a rat model of focal cerebral ischemia.

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.

Organic Nanoparticles with Aggregation-Induced Emission for Bone Marrow Stromal Cell Tracking in a Rat PTI Model.

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.

Effects of cerebral amyloid angiopathy on the brain vasculome.

ß-amyloid (Aß) deposits in brain blood vessel walls underlie the vascular pathology of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). Growing evidence has suggested the involvement of cerebrovascular dysfunction in the initiation and progression of cognitive impairment in AD and CAA patients. Therefore, in this study, we assessed the brain vasculome in a mouse model in order to identify cerebrovascular pathways that may be involved in AD and CAA vascular pathogenesis in the context of aging. Brain endothelial cells were isolated from young and old wild-type mice, and young and old transgenic mice expressing Swedish mutation in amyloid precursor protein and exon 9 deletion in presenilin 1 (APPswe/PSEN1dE9). Microarray profiling of these endothelial transcriptomes demonstrated that accumulation of vascular Aß in the aging APPswe/PSEN1dE9 mouse is associated with impaired endothelial expression of neurotransmitter receptors and calcium signaling transductors, while the genes involved in cell cycle and inflammation were upregulated. These results suggest that the vascular pathology of AD and CAA may involve the disruption of neurovascular coupling, reactivation of cell cycle in quiescent endothelial cells, and enhanced inflammation. Further dissection of these endothelial mechanisms may offer opportunities to pursue therapies to ameliorate vascular dysfunction in the aging brain of AD and CAA patients.

Early and Sustained Increases in Leukotriene B4 Levels Are Associated with Poor Clinical Outcome in Ischemic Stroke Patients.

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.

Absence of Stress Response in Dorsal Raphe Nucleus in Modulator of Apoptosis 1-Deficient Mice.

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.

Brain-to-cervical lymph node signaling after 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.

Promoting Neuro-Supportive Properties of Astrocytes with Epidermal Growth Factor Hydrogels.

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.

Modulator of apoptosis-1 is a potential therapeutic target in acute ischemic injury.

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.

Simultaneous functional photoacoustic microscopy and electrocorticography reveal the impact of rtPA on dynamic neurovascular functions after cerebral ischemia.

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.

Hydrogen sulfide in stroke: Protective or deleterious?

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.

Reprint of: Hydrogen sulfide in stroke: Protective or deleterious?

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.

Endogenous regeneration: Engineering growth factors for stroke.

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.

Integrated treatment modality of cathodal-transcranial direct current stimulation with peripheral sensory stimulation affords neuroprotection in a rat stroke model.

Cathodal-transcranial direct current stimulation induces therapeutic effects in animal ischemia models by preventing the expansion of ischemic injury during the hyperacute phase of ischemia. However, its efficacy is limited by an accompanying decrease in cerebral blood flow. On the other hand, peripheral sensory stimulation can increase blood flow to specific brain areas resulting in rescue of neurovascular functions from ischemic damage. Therefore, the two modalities appear to complement each other to form an integrated treatment modality. Our results showed that hemodynamics was improved in a photothrombotic ischemia model, as cerebral blood volume and hemoglobin oxygen saturation ([Formula: see text]) recovered to 71% and 76% of the baseline values, respectively. Furthermore, neural activities, including somatosensory-evoked potentials (110% increase), the alpha-to-delta ratio (27% increase), and the [Formula: see text] ratio (27% decrease), were also restored. Infarct volume was reduced by 50% with a 2-fold preservation in the number of neurons and a 6-fold reduction in the number of active microglia in the infarct region compared with the untreated group. Grip strength was also better preserved (28% higher) compared with the untreated group. Overall, this nonpharmacological, nonintrusive approach could be prospectively developed into a clinical treatment modality.

"Zipped Synthesis" by Cross-Metathesis Provides a Cystathionine ß-Synthase Inhibitor that Attenuates Cellular H2S Levels and Reduces Neuronal Infarction in a Rat Ischemic Stroke Model.

The gaseous neuromodulator H2S is associated with neuronal cell death pursuant to cerebral ischemia. As cystathionine ß-synthase (CBS) is the primary mediator of H2S biogenesis in the brain, it has emerged as a potential target for the treatment of stroke. Herein, a "zipped" approach by alkene cross-metathesis into CBS inhibitor candidate synthesis is demonstrated. The inhibitors are modeled after the pseudo-C 2-symmetric CBS product (l,l)-cystathionine. The "zipped" concept means only half of the inhibitor needs be constructed; the two halves are then fused by olefin cross-metathesis. Inhibitor design is also mechanism-based, exploiting the favorable kinetics associated with hydrazine-imine interchange as opposed to the usual imine-imine interchange. It is demonstrated that the most potent "zipped" inhibitor 6S reduces H2S production in SH-SY5Y cells overexpressing CBS, thereby reducing cell death. Most importantly, CBS inhibitor 6S dramatically reduces infarct volume (1 h post-stroke treatment; ∼70% reduction) in a rat transient middle cerebral artery occlusion model for ischemia.

Cystathionine ß-synthase inhibition is a potential therapeutic approach to treatment of ischemic injury.

Hydrogen sulfide (H2S) has been reported to exacerbate stroke outcome in experimental models. Cystathionine ß-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 with aggregation-induced emission for tracking bone marrow stromal cells in the rat ischemic stroke model.

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 attenuates interleukin-1ß-stimulated upregulation of chemokine CCL5 and glial fibrillary acidic protein in astrocytes.

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 1ß 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 1ß stimulation. These data suggest that andrographolide should be evaluated further as a therapeutic for central nervous system diseases characterized by astrocyte-mediated neuroinflammatory processes.