Yaying Song

Increased Circulating Exosomal miRNA-223 Is Associated with Acute Ischemic Stroke

Recent studies have demonstrated that exosomal microRNAs (miRNAs) are novel biomarkers and therapeutic targets for various diseases including vascular disease. However, specific exosomal miRNAs expression in stroke patients has not been reported yet. Here, we explored whether circulating exosomal miRNAs can serve as potential biomarkers for the diagnosis of acute ischemic stroke and discussed the potential for clinical application. Blood samples were collected from acute ischemic stroke patients within the first 72 h (n = 50). Circulating exosomes were exacted by Exoquick exosome isolation kit and characterized by transmission electron microscopy. Western blot was performed to assess the expression of exosomal protein makers. Exosomal miRNA-223 (miR-223) was detected by RT-PCR assay. The relationship between the expression levels of miR-223 and National Institutes of Health Stroke Scale (NIHSS) scores, brain infarct volume, and neurological outcomes were analyzed. Circulating exosomes were isolated and the size of vesicles ranged between 30 and 100 nm. The identification of exosomes was further confirmed by the detection of specific exosomal protein markers CD9, CD63, and Tsg101. Exosomal miR-223 in acute ischemic stroke patients was significantly upregulated compared to control group (p < 0.001). Exosomal miR-223 level was positively correlated with NIHSS scores (r = 0.31, p = 0.03). Exosomal miR-223 expression in stroke patients with poor outcomes was higher than those with good outcomes (p < 0.05). Increased exosomal miR-223 was associated with acute ischemic stroke occurrence, stroke severity, and short-term outcomes. Future studies with large sample are needed to assess the clinical application of exosomal miR-223 as a novel biomarker for ischemic stroke diagnosis.

Optogenetic Inhibition of Striatal Neuronal Activity Improves the Survival of Transplanted Neural Stem Cells and Neurological Outcomes after Ischemic Stroke in Mice

Neural stem cell (NSC) transplantation is a promising treatment to improve the recovery after brain ischemia. However, how the survival, proliferation, migration, and differentiation of implanted NSC are influenced by endogenous neuronal activity remains unclear. In this work, we used optogenetic techniques to control the activity of striatal neurons and investigated how their activity affected the survival and migration of transplanted NSCs and overall neurological outcome after ischemic stroke. NSCs cultured from transgenic mice expressing fluorescent protein were transplanted into the peri-infarct region of the striatum after transient middle cerebral artery occlusion (tMCAO) surgery. The striatal neurons were excited or inhibited for 15 minutes daily via implanted optical fiber after tMCAO. The results revealed that mice which received NSC transplantation and optogenetic inhibition had smaller brain infarct volume and increased NSC migration compared to the NSC alone or PBS group (p < 0.05). In contrast, mice which received NSC transplantation and optogenetic excitation showed no difference in infarct volume and neurological behavior improvement compared to the PBS control group. In vitro experiments further revealed that the conditioned media from excited GABAergic neurons reduced NSC viability through paracrine mechanisms. Conclusion. Optogenetic inhibition of striatal neuronal activity further improved neurological recovery after NSC transplantation at the subacute phase after brain ischemia.

CLARITY for High-resolution Imaging and Quantification of Vasculature in the Whole Mouse Brain

Elucidating the normal structure and distribution of cerebral vascular system is fundamental for understanding its function. However, studies on visualization and whole-brain quantification of vasculature with cellular resolution are limited. Here, we explored the structure of vasculature at the whole-brain level using the newly developed CLARITY technique. Adult male C57BL/6J mice undergoing transient middle cerebral artery occlusion and Tie2-RFP transgenic mice were used. Whole mouse brains were extracted for CLARITY processing. Immunostaining was performed to label vessels. Customized MATLAB code was used for image processing and quantification. Three-dimensional images were visualized using the Vaa3D software. Our results showed that whole mouse brain became transparent using the CLARITY method. Three-dimensional imaging and visualization of vasculature were achieved at the whole-brain level with a 1-μm voxel resolution. The quantitative results showed that the fractional vascular volume was 0.018 ± 0.004 mm3 per mm3, the normalized vascular length was 0.44 ± 0.04 m per mm3, and the mean diameter of the microvessels was 4.25 ± 0.08 μm. Furthermore, a decrease in the fractional vascular volume and a decrease in the normalized vascular length were found in the penumbra of ischemic mice compared to controls (p < 0.05). In conclusion, CLARITY provides a novel approach for mapping vasculature in the whole mouse brain at cellular resolution. CLARITY-optimized algorithms facilitate the assessment of structural change in vasculature after brain injury.

Optogenetic Inhibition of Striatal GABAergic Neuronal Activity Improves Outcomes After Ischemic Brain Injury

Background and Purpose— Striatal GABAergic neuron is known as a key regulator in adult neurogenesis. However, the specific role of striatal GABAergic neuronal activity in the promotion of neurological recovery after ischemic stroke remains unknown. Here, we used optogenetic approach to investigate these effects and mechanism. Methods— Laser stimulation was delivered via an implanted optical fiber to inhibit or activate the striatal GABAergic neurons in Gad2-Arch-GFP or Gad2-ChR2-tdTomato mice (n=80) 1 week after 60-minute transient middle cerebral artery occlusion. Neurological severity score, brain atrophy volume, microvessel density, and cell morphological changes were examined using immunohistochemistry. Gene expression and protein levels of related growth factors were further examined using real-time polymerase chain reaction and Western blotting. Results— Inhibiting striatal GABAergic neuronal activity improved functional recovery, reduced brain atrophy volume, and prohibited cell death compared with the control (P<0.05). Microvessel density and bFGF (basic fibroblast growth factor) expression in the inhibition group were also increased (P<0.05). In contrast, activation of striatal GABAergic neurons resulted in adverse effects compared with the control (P<0.05). Using cocultures of GABAergic neurons, astrocytes, and endothelial cells, we further demonstrated that the photoinhibition of GABAergic neuronal activity could upregulate bFGF expression in endothelial cells, depending on the presence of astrocytes. The conditioned medium from the aforementioned photoinhibited 3-cell coculture system protected cells from oxygen glucose deprivation injury. Conclusions— After ischemic stroke, optogenetic inhibition of GABAergic neurons upregulated bFGF expression by endothelial cells and promoted neurobehavioral recovery, possibly orchestrated by astrocytes. Optogenetically inhibiting neuronal activity provides a novel approach to promote neurological recovery.

cxcl12 Gene Engineered Endothelial Progenitor Cells Further Improve the Functions of Oligodendrocyte Precursor Cells

ABSTRACT Oligodendrocyte precursor cells (OPCs) are needed for white matter repair after various brain injury. Means that promote OPC functions could benefit white matter recovery after injury. Chemokine CXCL12 and endothelial progenitor cells (EPCs) both have been shown to promote remyelination. We hypothesize that the beneficial effects of EPCs and CXCL12 can be harnessed by genetically modifying EPCs with cxcl12 to synergistically improve the functions of OPCs. In this work, CXCL12‐EPC was generated using virus‐mediated gene transfer. OPCs were cultured with CXCL12‐EPC conditioned media (CM) to analyze its impact on the proliferation, migration, differentiation and survival properties of OPCs. We blocked or knocked‐down the receptors of CXCL12, namely CXCR4 and CXCR7, respectively to investigate their functions in regulating OPCs properties. Results revealed that CXCL12‐EPC CM further promoted OPCs behavioral properties and upregulated the expression of PDGFR‐&agr;, bFGF, CXCR4 and CXCR7 in OPCs, albeit following different time course. Blocking CXCR4 diminished the beneficial effects of CXCL12 on OPCs proliferation and migration, while knocking down CXCR7 inhibited OPCs differentiation. Our results supported that cxcl12 gene modification of EPCs further promoted EPCs’ ability in augmenting the remyelination properties of OPCs, suggesting that CXCL12‐EPC hold great potential in white matter repair. HIGHLIGHTScxcl12 gene engineering of EPCs promoted VEGF and IGF expression by EPCs.Conditioned media from CXCL12‐EPCs further promoted the functions of OPCs.CXCL12‐EPCs conditioned media affected the expression of trophic factors by OPCs.CXCR4 knockdown suppressed the proliferation and migration of OPCs and reduced PDGFR‐&agr; and bFGF expression.CXCR7 knockdown inhibited the expression of MBP by OPCs and caused slower differentiation.

Blood-Brain Barrier Disruption Induced Cognitive Impairment Is Associated With Increase of Inflammatory Cytokine

Patients with diabetes suffer the higher risk of dementia and the underlying pathological mechanism of cognitive dysfunction in diabetes is not fully understood. In this study, we explore whether the cognitive impairment in the diabetic rat is associated with increased blood brain barrier (BBB) permeability and the change of the inflammatory cytokine. Experimental diabetic rats were induced by single intraperitoneal injection of streptozotocin (STZ). Cognitive function was evaluated by Morris water maze in the normal and the diabetic rats, respectively. The spatial acquisition trials were conducted over five consecutive days and the probe test was performed on day 6, followed by working memory test on the next 4 days. Escape latency was recorded in the acquisition trials and working memory test; time spent in the target quadrant and the number of crossing the former platform were recorded in the probe test. BBB permeability was assessed by measuring the extravasation of IgG. The image of occludin and claudin-5 staining by a confocal microscope were acquired to measure the gap in the tight junction. Cytokines TNF-α, IL-1β and IL-6 mRNA expression were further examined by Real-time PCR. The time spent in the target quadrant within 30 s decreased in the 8-week STZ rats compared to that of the normal rats (p < 0.05), while no difference was seen in the performance of working memory between the diabetic and normal rats. IgG leakage significantly increased in the brain parenchyma of the 8-week STZ rats compared to the normal rats (p < 0.05). The immunostaining of occludin and claudin-5 suggested the gap in the tight junction increased in the 8-week STZ rats compared to the normal rats (p < 0.05). Moreover, TNF-α and IL-6 mRNA also increased in the brain of 8-week STZ rats compared to the normal rats (p < 0.05). These results suggested that loss of BBB integrity might contribute to progressive impairment of cognitive in the diabetic rats. The increase of TNF-α and IL-6 expression might trigger the disruption of BBB in the brain, which eventually caused cognitive impairment in the 8-week STZ rats.

Stem Cell Therapy in Stroke

Stem cell-based treatment for ischemic stroke has shown its effectiveness in animal models and clinical trials. In this chapter, we describe different types and delivery routes of stem cells for therapy, the tracing of stem cells after delivery, and the clinical challenges and strategies in the future. Stem cells derived from various tissues have shown their beneficial role for functional recovery after stroke. Although the mechanism of stem cell-based therapy is not fully understood, it may include the releasing of growth factors, microenvironment regulation, and the preparation of repairing the blood-brain barrier integrity. Clinical applications of stem cell-based therapy are still in infancy. The future of clinical study in the stem cell-based therapy in the stroke field needs to focus on the modification of stem cells or combining different types of stem cells to enhance the therapeutic efficacy, mechanisms of stem cells action, and translation to clinical applications. Stem cell treatment is a promising regenerative therapeutic strategy because it can prevent neuronal cell apoptosis, inhibit pro-inflammatory cell recruitment, secrete multiple neurotropic factors, and promote neural differentiation.

MicroRNA‐137 and ‐195* inhibit vasculogenesis in brain arteriovenous malformations

Brain arteriovenous malformations (AVMs) are the most common cause of nontraumatic intracerebral hemorrhage in young adults. The genesis of brain AVM remains enigmatic. We investigated microRNA (miRNA) expression and its contribution to the pathogenesis of brain AVMs.