Our previous study indicated that nicotinamide phosphoribosyltransferase (NAMPT) is released from cells and might be an important extracellular neuroprotective factor in brain ischemia. Here, we tested whether NAMPT protects against ischemic brain injury when administered directly into the intracerebroventricular (ICV) compartment of the cranium. Recombinant NAMPT protein (2 μg) was delivered ICV in mice subjected to 45-min middle cerebral artery occlusion (MCAO), and the effects on infarct volume, sensorimotor function, microglia/macrophage polarization, neutrophil infiltration, and BBB integrity were analyzed. The results indicate that ICV administration of NAMPT significantly reduced infarct volume, retained its beneficial properties even when ICV administration was delayed by 6 h after MCAO, and improved neurological outcomes. NAMPT treatment inhibited pro-inflammatory microglia/macrophages, promoted microglia/macrophage polarization toward the anti-inflammatory phenotype, and reduced the infiltration of neutrophils into the perilesional area after brain ischemia. In vitro studies indicated that multiple pro-inflammatory microglial markers/cytokines were downregulated while multiple anti-inflammatory microglial markers/cytokines were induced in primary microglial cultures treated with NAMPT protein. NAMPT treatment also fortified the blood–brain barrier (BBB), as shown by reduced extravascular leakage of the small-molecule tracer Alexa Fluor 555 Cadaverine and larger-sized endogenous IgGs into brain parenchyma. Thus, NAMPT may protect against ischemic brain injury partly through a novel anti-inflammatory mechanism, which in turn maintains BBB integrity and reduces the infiltration of peripheral inflammatory cells. Taken together, these results provide validation of recombinant NAMPT delivery into the extracellular space as a potential neuroprotective strategy for stroke.
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This project was supported by National Institutes of Health/NINDS grants NS079345 (to G.C.) and Department of Veterans Affairs Merit Review grants BX002346 and BX003923 (to G.C.). J.X. was supported by National Natural Science Foundation of China (81722028 and 81572237) and Natural Science Foundation of Zhejiang Province (R18H50001).
Compliance with Ethical Standards
All animal procedures used in this study were conducted in strict compliance with The National Institue of Health Guide for Use and Care of Laboratory Animals and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplemental Table 1Animal number/group, animal exclusions and mortality for the entire project. Detailed information for animal number/group, the numbers that died, and reasons that animals died or were excluded from the experiment. (DOCX 29.1 kb)
Effect of LPS contamination in recombinant NAMPT protein on infarct volumes after MCAO. Mice were subjected to MCAO and treated with ICV injections of NAMPT with or without LPS removal, and infarct volumes were determined with TTC staining 72 h after MCAO. PBS was used as the vehicle treatment control. Quantitative analysis showed that infarct reduction offered by NAMPT protein was negated by LPS contamination. ***p < 0.001 versus PBS treatment, ###p < 0.001 versus without LPS removal. N = 8, 9, 8 for PBS, NAMPT with or without LPS removal. (PNG 55 kb)
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