Targeting stroke-specific brain glycan signature using engineered biomaterials

Introduction: Stroke is a leading cause of adult disability in the United States and lacks effective treatment. Aging is the most potent non-modifiable risk factor for stroke, the incidence of which doubles every 10 years above age 55 (1). With the projected increase in the number of people over 65 in the next decade (2), there is an expected rise in stroke cases (3). Glycosylation is critically implicated in multiple physiological and pathological processes including central nervous system (CNS) disorders. An ischemic stroke is a sudden blood flow blockage in the brain, which induces neuroinflammation, and leads to permanent tissue damage and long-term physical disability. Although previous studies have implied the significance of glycan changes in neuroinflammation, changes in tissue glycosylation patterns during ischemic stroke or after treatment have not been investigated. We hypothesize that our pro-angiogenic therapeutic biomaterial intervention attenuates stroke glycosignature, leading to the identification of potent glycosylation targets for brain regeneration and motivating an optimal design of future glycan-functionalized biomaterials with greater therapeutic impact. The current study reports a novel, stroke-specific glycan marker and its modulation following a therapeutic biomaterial treatment in ischemic stroke to identify the glycosylation cues involved in tissue repair.

Materials and

Methods: To study the spatial modulation of brain glycosignature from the in-vivo time course analysis of ischemic stroke progression in a rodent photothrombotic stroke model and following therapeutic biomaterial treatment (4), we developed and used an elaborate multi-modal glycoanalytical platform. Modulation of gene expression for the glycosylation enzymes over one month was investigated using real-time polymerase chain reaction (RT-PCR). Tissue-level modulation of overall glycosylation was represented using lectin histochemistry. Corresponding quantification and spatial resolution of glycan modulation in the tissue was performed using Matrix-assisted laser desorption electrospray ionization mass spectrometric imaging (IR-MALDESI MSI). An Orbitrap Exploris 240 mass spectrometer was used for data collection for MSI in negative ionization mode within a range of 500 to 2000 m/z.

Results, Conclusions, and Discussions: To the best of our knowledge, this is the first investigation of brain tissue glycosignature in stroke and following biomaterial treatment. Using IR-MALDESI analysis of stroke brains, we report a novel stroke-specific glycan marker that shows minimal detection in healthy brains. The current study also investigates the mechanism of development of this stroke-specific glycan marker and its attenuation using the pro-angiogenic therapeutic material. The results from the temporal analysis of glycosylation enzymes and tissue-level glycan expression showed an overall reduction in sialylation and complex glycosylation and an increase in fucosylation and mannosylation. A concomitant change in tissue glycosylation was observed using lectin histochemistry. These trends of ‘injury-specific’ tissue glycosylation in ischemic stroke are remarkably reversed by the application of a previously developed pro-regenerative therapeutic biomaterial (4). The results show the involvement of glycan-mediated pathways in brain repair observed as a result of the application of engineered materials.

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