Exosomes derived from bone marrow mesenchymal stem cells improve the integrity of the blood-spinal cord barrier after spinal cord injury

doi:10.12307/2022.134

Abstract

Abstract: BACKGROUND: The studies reveal that stem cells-derived exosomes promote locomotor recovery after spinal cord injury.
OBJECTIVE: To investigate whether the bone marrow mesenchymal stem cells-derived exosomes promote locomotor recovery via inhibiting the damage of the blood-spinal cord barrier.
METHODS: Sixty Sprague-Dawley rats were randomly divided into sham operation group, model group, and exosome group (n=20). The rat models of spinal cord injury were established by modified Allen’s method. In the exosome group, 200 μL of bone marrow mesenchymal stem cells-derived exosomes were injected through the tail vein at 30 minutes and 1 day after injury. On the third day after injury, the permeability of the blood-spinal cord barrier was observed. Western blot assay was used to detect the expression of matrix metalloproteinase-9 and claudin-5, Occludin and ZO-1. Gelatin zymography was used to detect the activity of matrix metalloproteinase-9. Immunofluorescence was used to detect the infiltration of neutrophils. Hematoxylin-eosin staining was used to observe the morphological changes of spinal cord injury. At 1, 3, 5, 7, 10, 14, and 21 days after injury, the Basso-Beattie-Bresnahan rating scale was used to evaluate the recovery of motor function.
RESULTS AND CONCLUSION: (1) The Basso-Beattie-Bresnahan scores of rats treated with exosome were significantly higher than these spinal cord injury rats at 10, 14, and 21 days after spinal cord injury (P < 0.05). The results of hematoxylin-eosin staining showed that the damage area in the exosome group was significantly reduced compared to the model group (P < 0.05). (2) Exosome treatment significantly reduced the amount of Evans Blue dye extravasation (P < 0.05). Compared with the model group, the expression levels of tight junction protein, including claudin-5, Occludin and ZO-1, were significantly increased in the exosome group (P < 0.05). (3) Exosome inhibited matrix metalloproteinase-9 expression and activity (P < 0.05). (4) Infiltrated myeloperoxidase-positive neutrophils were observed at the lesion site of injured spinal cord at 3 days after spinal cord injury. The exosome treatment significantly inhibited the infiltration of neutrophils (P < 0.05). (5) The results suggested that bone marrow mesenchymal stem cells-derived exosome improved functional recovery after spinal cord injury in part by inhibiting blood-spinal cord barrier disruption and the subsequent infiltration of neutrophils through reducing the degradation of tight junction proteins by inhibiting the expression and activity of matrix metalloproteinase-9.

Key words: stem cell, bone marrow mesenchymal stem cell, exosome, spinal cord injury, blood-spinal cord barrier, matrix metalloprotease-9, tight junction protein, neutrophil

CLC Number:

Hu Wei, Xie Xingqi, Tu Guanjun. Exosomes derived from bone marrow mesenchymal stem cells improve the integrity of the blood-spinal cord barrier after spinal cord injury[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(7): 992-998.

Figures/Tables 5

References

[1] YILMAZ T, KAPTANOĞLU E. Current and future medical therapeutic strategies for the functional repair of spinal cord injury. World J Orthop. 2015;6(1):42-55.
[2] PARK CS, LEE JY, CHOI HY, et al. Protocatechuic acid improves functional recovery after spinal cord injury by attenuating blood-spinal cord barrier disruption and hemorrhage in rats. Neurochem Int. 2019;124: 181-192.
[3] BARTANUSZ V, JEZOVA D, ALAJAJIAN B, et al. The blood-spinal cord barrier: morphology and clinical implications. Ann Neurol. 2011;70(2): 194-206.
[4] FAN B, WEI Z, YAO X, et al. Microenvironment Imbalance of Spinal Cord Injury. Cell Transplant. 2018;27(6):853-866.
[5] FIGLEY SA, KHOSRAVI R, LEGASTO JM, et al. Characterization of vascular disruption and blood-spinal cord barrier permeability following traumatic spinal cord injury. J Neurotrauma. 2014;31(6):541-552.
[6] ZLOKOVIC BV. The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron. 2008;57(2):178-201.
[7] LEE JY, CHOI HY, YUNE TY. Fluoxetine and vitamin C synergistically inhibits blood-spinal cord barrier disruption and improves functional recovery after spinal cord injury. Neuropharmacology. 2016;109:78-87.
[8] MARTINS T, BAPTISTA S, GONÇALVES J, et al. Methamphetamine transiently increases the blood-brain barrier permeability in the hippocampus: role of tight junction proteins and matrix metalloproteinase-9. Brain Res. 2011;1411:28-40.
[9] GONG Z, XIA K, XU A, et al. Stem Cell Transplantation: A Promising Therapy for Spinal Cord Injury. Curr Stem Cell Res Ther. 2020;15(4): 321-331.
[10] ROMANELLI P, BIELER L, SCHARLER C, et al. Extracellular Vesicles Can Deliver Anti-inflammatory and Anti-scarring Activities of Mesenchymal Stromal Cells After Spinal Cord Injury. Front Neurol. 2019;10:1225.
[11] JEONG JO, HAN JW, KIM JM, et al. Malignant tumor formation after transplantation of short-term cultured bone marrow mesenchymal stem cells in experimental myocardial infarction and diabetic neuropathy. Circ Res. 2011;108(11):1340-1347.
[12] YUAN X, WU Q, WANG P, et al. Exosomes Derived From Pericytes Improve Microcirculation and Protect Blood-Spinal Cord Barrier After Spinal Cord Injury in Mice. Front Neurosci. 2019;13:319.
[13] KYURKCHIEV D, BOCHEV I, IVANOVA-TODOROVA E, et al. Secretion of immunoregulatory cytokines by mesenchymal stem cells. World J Stem Cells. 2014;6(5):552-570.
[14] MAROTE A, TEIXEIRA FG, MENDES-PINHEIRO B, et al. MSCs-Derived Exosomes: Cell-Secreted Nanovesicles with Regenerative Potential. Front Pharmacol. 2016;7:231.
[15] WIKLANDER OPB, BRENNAN MÁ, LÖTVALL J, et al. Advances in therapeutic applications of extracellular vesicles. Sci Transl Med. 2019;11(492):eaav8521.
[16] KALLURI R, LEBLEU VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367(6478):eaau6977.
[17] SHAO J, ZARO J, SHEN Y. Advances in Exosome-Based Drug Delivery and Tumor Targeting: From Tissue Distribution to Intracellular Fate. Int J Nanomedicine. 2020;15:9355-9371.
[18] GURUNATHAN S, KANG MH, JEYARAJ M, et al. Review of the Isolation, Characterization, Biological Function, and Multifarious Therapeutic Approaches of Exosomes. Cells. 2019;8(4):307.
[19] KORNILOV R, PUHKA M, MANNERSTRÖM B, et al. Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. J Extracell Vesicles. 2018;7(1):1422674.
[20] RONG Y, LIU W, LV C, et al. Neural stem cell small extracellular vesicle-based delivery of 14-3-3t reduces apoptosis and neuroinflammation following traumatic spinal cord injury by enhancing autophagy by targeting Beclin-1. Aging (Albany NY). 2019;11(18):7723-7745.
[21] WANG L, PEI S, HAN L, et al. Mesenchymal Stem Cell-Derived Exosomes Reduce A1 Astrocytes via Downregulation of Phosphorylated NFκB P65 Subunit in Spinal Cord Injury. Cell Physiol Biochem. 2018;50(4): 1535-1559.
[22] BASSO DM, BEATTIE MS, BRESNAHAN JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 1995;12(1):1-21.
[23] WU P, ZHANG B, OCANSEY DKW, et al. Extracellular vesicles: A bright star of nanomedicine. Biomaterials. 2021;269:120467.
[24] MENG W, HE C, HAO Y, et al. Prospects and challenges of extracellular vesicle-based drug delivery system: considering cell source. Drug Deliv. 2020; 27(1):585-598.
[25] KJELL J, OLSON L. Rat models of spinal cord injury: from pathology to potential therapies. Dis Model Mech. 2016;9(10):1125-1137.
[26] YU D, SUN R, SHEN D, et al. Nuclear heme oxygenase-1 improved the hypoxia-mediated dysfunction of blood-spinal cord barrier via the miR-181c-5p/SOX5 signaling pathway. Neuroreport. 2021;32(2):112-120.
[27] REINHOLD AK, RITTNER HL. Barrier function in the peripheral and central nervous system-a review. Pflugers Arch. 2017;469(1):123-134.
[28] KUMAR H, ROPPER AE, LEE SH, et al. Propitious Therapeutic Modulators to Prevent Blood-Spinal Cord Barrier Disruption in Spinal Cord Injury. Mol Neurobiol. 2017;54(5):3578-3590.
[29] KUMAR H, JO MJ, CHOI H, et al. Matrix Metalloproteinase-8 Inhibition Prevents Disruption of Blood-Spinal Cord Barrier and Attenuates Inflammation in Rat Model of Spinal Cord Injury. Mol Neurobiol. 2018; 55(3):2577-2590.
[30] SHENDE P, SUBEDI M. Pathophysiology, mechanisms and applications of mesenchymal stem cells for the treatment of spinal cord injury. Biomed Pharmacother. 2017;91:693-706.
[31] MUKHAMEDSHINA YO, GRACHEVA OA, MUKHUTDINOVA DM, et al. Mesenchymal stem cells and the neuronal microenvironment in the area of spinal cord injury. Neural Regen Res. 2019;14(2):227-237.
[32] HA XQ, YANG B, HOU HJ, et al. Protective effect of rhodioloside and bone marrow mesenchymal stem cells infected with HIF-1-expressing adenovirus on acute spinal cord injury. Neural Regen Res. 2020;15(4):690-696.
[33] ZHOU Z, CHEN Y, ZHANG H, et al. Comparison of mesenchymal stromal cells from human bone marrow and adipose tissue for the treatment of spinal cord injury. Cytotherapy. 2013;15(4):434-448.
[34] YANG Y, YE Y, SU X, et al. MSCs-Derived Exosomes and Neuroinflammation, Neurogenesis and Therapy of Traumatic Brain Injury. Front Cell Neurosci. 2017;11:55.
[35] XU G, AO R, ZHI Z, et al. miR-21 and miR-19b delivered by hMSC-derived EVs regulate the apoptosis and differentiation of neurons in patients with spinal cord injury. J Cell Physiol. 2019;234(7):10205-10217.
[36] HUANG JH, YIN XM, XU Y, et al. Systemic Administration of Exosomes Released from Mesenchymal Stromal Cells Attenuates Apoptosis, Inflammation, and Promotes Angiogenesis after Spinal Cord Injury in Rats. J Neurotrauma. 2017;34(24):3388-3396.
[37] JIANG D, GONG F, GE X, et al. Neuron-derived exosomes-transmitted miR-124-3p protect traumatically injured spinal cord by suppressing the activation of neurotoxic microglia and astrocytes. J Nanobiotechnology. 2020;18(1):105.
[38] RUPPERT KA, NGUYEN TT, PRABHAKARA KS, et al. Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Modify Microglial Response and Improve Clinical Outcomes in Experimental Spinal Cord Injury. Sci Rep. 2018;8(1):480.
[39] HUANG JH, XU Y, YIN XM, et al. Exosomes Derived from miR-126-modified MSCs Promote Angiogenesis and Neurogenesis and Attenuate Apoptosis after Spinal Cord Injury in Rats. Neuroscience. 2020;424: 133-145.
[40] LU Y, ZHOU Y, ZHANG R, et al. Bone Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Recovery Following Spinal Cord Injury via Improvement of the Integrity of the Blood-Spinal Cord Barrier. Front Neurosci. 2019;13:209.
[41] STERNLICHT MD, WERB Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol. 2001;17:463-516.
[42] LEE JY, CHOI HY, PARK CS, et al. Mithramycin A Improves Functional Recovery by Inhibiting BSCB Disruption and Hemorrhage after Spinal Cord Injury. J Neurotrauma. 2018;35(3):508-520.
[43] NOBLE LJ, DONOVAN F, IGARASHI T, et al. Matrix metalloproteinases limit functional recovery after spinal cord injury by modulation of early vascular events. J Neurosci. 2002;22(17):7526-7535.
[44] LEE JY, CHOI HY, NA WH, et al. Ghrelin inhibits BSCB disruption/hemorrhage by attenuating MMP-9 and SUR1/TrpM4 expression and activation after spinal cord injury. Biochim Biophys Acta. 2014;1842 (12 Pt A):2403-2412.
[45] BECK KD, NGUYEN HX, GALVAN MD, et al. Quantitative analysis of cellular inflammation after traumatic spinal cord injury: evidence for a multiphasic inflammatory response in the acute to chronic environment. Brain. 2010;133(Pt 2):433-447.
[46] YAO Y, XU J, YU T, et al. Flufenamic acid inhibits secondary hemorrhage and BSCB disruption after spinal cord injury. Theranostics. 2018;8(15): 4181-4198.
[47] SAIWAI H, OHKAWA Y, YAMADA H, et al. The LTB4-BLT1 axis mediates neutrophil infiltration and secondary injury in experimental spinal cord injury. Am J Pathol. 2010;176(5):2352-2366.