Evaluating the efficacy of neural stem cells combined with Reelin-loaded PLGA-PEG micelles in regeneration of infract cavity and improvement of neurological function in photothrombotic stroke model of mice

Abstract

Abstract Introduction: Ischemic stroke is characterized by extensive neuronal loss, glial scar formation, neural tissue degeneration which leading to profound changes in the extracellular matrix, neuronal circuitry, and long-lasting functional disabilities. Although transplanted neural stem cells (NSCs) can recover some of the functional deficit after stroke, retrieval is not complete and repair of lost tissue is negligible. Therefore, the current challenge is to use the combination of NSCs with suitably enriched biomaterials to retain these cells within the infarct cavity and accelerate the formation of a de novo tissue. This study aimed to test the regenerative potential of PLGA-PEG micelle biomaterial enriched with Reelin and embryonic NSCs on photochemical stroke model of mice to gain appropriate method in tissue engineering. Material and methods: For this purpose, two sets of experiments, either in vitro or in vivo experiment, were performed. This study tested the in vitro effect of PLGA-PEG micelle, Reelin, and Reelin-loaded PLGA-PEG on embryonic NSCs proliferation, differentiation, and neurite growth. In in vivo experiment, stroke damaged mice were transplanted with PBS, NSCs, PLGA-PEG+NSCs, Reelin+NSCs, and Reelin-loaded PLGA-PEG+NSCs and the migration and differentiation of NSCs were evaluated. Immunohistochemistry analysis used for assessment of astrocytic gliosis and local angiogenesis in the periphery of lesion site. Additionally, the cavity size was measured using stereological study and the mNSS test was performed to assess neurological function. Results: In vitro assays exhibited PLGA-PEG plus Reelin-induced proliferation rate (Ki-67+ NSCs) and neurite outgrowth (axonization and dendritization) compared to PLGA-PEG+NSCs and Reelin+NSCs groups (p<0.05). Besides, neural differentiation (Map-2+ cells) was high in NSCs cultured on PLGA-PEG+ Reelin (p<0.05). Double immunofluorescence staining showed that Reelin-loaded PLGA-PEG increased the number of neural progenitor cells (DCX) and mature neurons (NeuN) around the lesion site compared to the groups received PLGA-PEG and Reelin alone after one month (p<0.05). Immunohistochemistry results showed that the PLGA/PEG plus Reelin significantly decreased the astrocytic gliosis and increased local angiogenesis (vWF-positive cells) relative to the other groups. These changes led to the reduction of cavity size in the Reelin-loaded PLGA-PEG+NSCs group. Neurobehavioral tests indicated Reelin-loaded PLGA-PEG+NSCs promoted neurological outcome and functional recovery (p<0.05). conclusion: These results indicated that Reelin-loaded PLGA-PEG is capable of promoting NSCs dynamic growth, neuronal differentiation, and local angiogenesis following ischemic injury via providing a desirable microenvironment. These features can lead to neural tissue regeneration and functional recovery