Introduction: Ischemic stroke, a leading cause of disability, is caused by occlusion of blood flow in the brain, resulting in inflammation, breakdown of the blood-brain-barrier, tissue necrosis, and the formation of the stroke infarct, a fluid-filled cavity. The introduction of microporous annealed particle (MAP) hydrogels into this cavity in the subacute phase of injury improves regenerative outcomes. A concurrent change occurs in the immune response: MAP hydrogels reduce inflammation and astrocyte reactivity1-3(Fig.A,B), decrease macrophage/microglica presence in peri-infarct2, 3(Fig.A,C), and promote pro-reparative phenotypes in the infarct3(Fig.D,E). These findings indicate MAP hydrogels may operate through an immunomodulatory mechanism. Further supporting this, MAP hydrogels functionalized with extracellular vesicles (EVs) derived from reactive-astrocytes, a known axis of communication with the peripheral immune system4, promote functional improvement5(Fig.F) and increase T-cell infiltration(Fig.G). However, the results discussed above are quantified from immunostaining; a full, purely-quantitative characterization of the immune changes induced by MAP hydrogels in the context of ischemic stroke has not been performed, particularly in terms phenotypic and temporal changes. Using spectral flow cytometry for high dimensional investigation, we have performed validation studies of immune characterization of the post-stroke brain(Fig.H), and we plan to extend these studies by profiling the immune response to MAP hydrogels, with and without functionalization of EVs, in evolution of ischemic stroke(Fig.I). This will improve our understanding of the neuro-immune environment following ischemic stroke and elucidate the immunomodulatory capacity of MAP hydrogels in the post-stroke brain, probing a possible mechanism of action for the increased regenerative potential observed with MAP hydrogel treatment.
Materials and
Methods: A photothrombotic mouse model of ischemic stroke is used for these studies. Young male (7-12 weeks) C57BL6 mice are injected intraperitoneally with Rose Bengal (10 mL/g), a photosensitive dye that leads to free radical formation and microvascular damage at the exposure of UV light. A midline incision exposes the skull, and a burr hole is drilled in the skull 1.5 mm laterally left of the bregma, overlying the motor cortex. This area is irradiated to induce vascular damage.
At Day 5 after stroke, the stroke cavity can accommodate 4-6 µL of MAP hydrogel; solutions of hyaluronic acid functionalized with tetrazine groups and hyaluronic acid microparticle hydrogels functionalized with norbornene groups are mixed and loaded into a Hamilton syringe, then injected through the burr hole, allowing the microparticles to anneal to form MAP hydrogel in situ via norbornene-tetrazine click-chemistry. MAP hydrogels are functionalized with EVs through azide-DBCO, bio-orthagonal, click-chemistry using EVs isolated from activated astrocytes which have been metabolically labeled with azides.
Animals are sacrificed on Day 7, 10, 14, and 21 and transcardially perfused with PBS to remove red blood cells. For immunostaining, this is followed with PFA perfusion, cryosection, and staining. For spectral flow cytometry, single cells are isolated from live brain tissue by enzymatic and mechanical digestion followed by density-gradient demyelination. T-cell (PD-1, RORyT, Gata3, CD25, CD45, CD3e, CD4, CD8a, FoxP3) and macrophage/microglia (CD206, CD86, TMEM119, CD11c, MHCII, CD45, iNOS, F4/80, Arginase 1, CD11b, IBA1, P2RY12, CX3CR1, TREM2) panels are evaluated by spectral flow cytometry for immunophenotyping.
Results, Conclusions, and Discussions: Immunostaining from previous studies of MAP hydrogels in the context of ischemic stroke has indicated a clear immune response to the biomaterial. In the presence of MAP hydrogels, reactive astrocytes establish a significantly thinner GFAP+ glial barrier2, 3(Fig.A,B) and the presence of Iba1+ microglia is significantly decreased2, 3(Fig.A,C), suggesting a less inflammatory environment. MAP hydrogels result in a significantly higher Arg1/DAPI ratio, suggesting a higher presence of pro-regenerative phenotypes3(Fig.D,E). The addition of reactive-astrocyte EVs to MAP hydrogels induces functional recovery5(Fig.F) and increases T-cell infiltration(Fig.G).
Initial spectral flow cytometry results validate that we are able to isolate cells from stroked brain tissue at high viability(Fig.H). Initial validation studies have confirmed the presence of immune cell markers in the brain using our photothrombotic stroke model(Fig.H). Using these methods, we will investigate the time course and phenotype of the immune response to MAP hydrogel biomaterials implanted intracranially at Day 5 post-stroke in comparison to untreated stroke and healthy animals. We also plan to include studies of the immune response to MAP functionalized with extracellular vesicles derived from activated astrocytes(Fig.I).
The spectral flow cytometry studies detailed herein will further characterize the immune response to MAP hydrogels by interrogating the timing and phenotype of immune response in detail. This work will further our understanding of the neuro-immune stroke environment, investigate immunomodulatory capacity of MAP hydrogels in stroke, and investigate a possible mechanism of action for MAP-based regeneration. As such, this work is highly significant in advancing our efforts toward implementing MAP hydrogels as a translational therapy to treat stroke-related disability.
Acknowledgements (Optional):
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Acknowledgements: This work was made possible by NSF GRFP and NIH.
