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.

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.

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.

"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.

Brain 3-Mercaptopyruvate Sulfurtransferase (3MST): Cellular Localization and Downregulation after Acute Stroke.

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 ß-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.

Neuroprotective effects of andrographolide in a rat model of permanent cerebral ischaemia.

BACKGROUND AND PURPOSE: Andrographolide is a diterpenoid lactone isolated from a traditional medicinal herb, Andrographis paniculata. It possesses potent anti-inflammatory activity. The present study examined potential therapeutic effects of andrographolide on cerebral ischaemia using a rat model with permanent middle cerebral artery occlusion (pMCAO). EXPERIMENTAL APPROACH: The MCA in rats was permanently occluded (by cautery), and 24 h later neurological effects were assessed with behavioural scores. Infarct volume and microglial activation were determined histologically. The p65 form of the transcription factor, nuclear factor-κB (NF-κB), was measured by Western blot, and cytokines by immunoassay of brain extracts. KEY RESULTS: Andrographolide, given i.p. 1 h after pMCAO, reduced infarct volume with a maximum reduction of approximately 50% obtained at 0.1 mg·kg(-1). Neurological deficits were also reduced by andrographolide, reflecting a correlation between infarct volume and neurological deficits. pMCAO was found to induce activation of microglia and elevate tumour necrosis factor (TNF)-α, interleukin (IL)-1ß and prostaglandin (PG)E(2) in the ischaemic brain areas. Andrographolide (0.1 mg·kg(-1)) significantly attenuated or abolished these effects. In addition, andrographolide suppressed the translocation of p65 from cytosol to nucleus, indicating reduced NF-κB activation. CONCLUSIONS AND IMPLICATIONS: Andrographolide exhibited neuroprotective effects, with accompanying suppression of NF-κB and microglial activation, and reduction in the production of cytokines including TNF-α and IL-1ß, and pro-inflammatory factors such as PGE(2). Our findings suggest that andrographolide may have therapeutic value in the treatment of stroke.