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Fig. 7 | Journal of Neuroinflammation

Fig. 7

From: Long noncoding RNA MALAT1 in exosomes drives regenerative function and modulates inflammation-linked networks following traumatic brain injury

Fig. 7

Localization of exosomes and validation of MALAT1 and GFP levels. Localization of exosomes and MALAT1 to the brain and spleen. Confocal imaging of exosome-positive expression of GFP in the brain and spleen of TBI rats. Images reveal migration of exosomes to both the contralateral and ipsilateral cortical region of the TBI-impacted brains near the impact site (a, b, d, e) and the spleen (c, f) of TE and TEdM rats as shown by detecting GFP expression (green) in cells with DAPI-positive staining (blue) 11 days after transplantation. White arrows indicate cells with exosomes. There was higher migration to the ipsilateral cortex near the impact site than that observed in the contralateral cortex which agrees with our ex vivo imaging data in Fig. 3. Scale bar in f equals 50 μm. g MALAT1 expression was measured in hASC-isolated exosomes and isolated exosomes with MALAT1 knockdown. Relative to the MALAT1 level in hASC, there is an increase in MALAT1 in the secreted exosomes, and this was knocked down significantly with the antisense oligonucleotide treatment. The results were analyzed with two-tailed Student’s t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of p < 0.05 was considered statistically significant. ***p < 0.001. h Endogenous MALAT1 levels in rat brain and spleen decrease with TBI: Following TBI and treatment with hASC exosomes, the levels of MALAT1 within the rat brain and spleen were analyzed by PCR using rat-specific primers. RNA was extracted and samples were pooled from four rats each from the no TBI, TBI treated with vehicle, and TBI treated with exosomes. Graph represents percent control with no TBI group designated as 100%. The experiment was repeated five times. The results were analyzed with two-tailed Student’s t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of p < 0.05 was considered statistically significant. ***p < 0.001 highly significant between control (no TBI) and TBI with vehicle treatment and between TBI and TBI treated with hASC exosomes for MALAT1. i Verification that hASC exosomes were taken up by the brain and spleen: SYBR Green real-time qPCR using human MALAT1 primers and j GFP primers was performed for absolute quantification. GAPDH served as control. For absolute quantification, a standard curve was generated for each gene in every assay. Absolute quantification of mRNA expression levels for MALAT1 and GFP was calculated by normalizing the values to GAPDH. The experiments were repeated four times with similar results. The results were analyzed with two-tailed Student’s t test using PRISM4 statistical analysis software (GraphPad, San Diego, CA). A level of p < 0.05 was considered statistically significant. ***p < 0.0001 highly significant between TBI treated with hASC exosomes and TBI treated with MALAT1-depleted (M1 aso) exosomes for MALAT1. The levels of GFP did not change with depletion of MALAT1

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