Near infrared light amplifies endothelial progenitor cell accumulation after stroke

Abstract

Damage-associated molecular pattern signals may play key roles in mediating non-cell autonomous effects of pre and post-conditioning. Here, we show that near-infrared (NIR) light stimulation of astrocytes increases a calcium-dependent secretion of the prototypical DAMP, HMGB1, which may then accelerate endothelial progenitor cell (EPC) accumulation after stroke. Conditioned media from NIR-stimulated astrocytes increased EPC proliferation in vitro, and blockade of HMGB1 with siRNA diminished the effect. In vivo transcranial NIR treatment confirmed that approximately 40% of NIR could penetrate the scalp and skull. Concomitantly, NIR increased GFAP expression in normal mouse brain at 30 min after the irradiation. In a mouse model of focal ischemia, repeated irradiation of NIR at days 5, 9, and 13 successfully increased HMGB1 in peri-infarct cortex, leading to a higher accumulation of EPCs at 14 days post-stroke. Conditioning and tolerance are now known to involve cell-cell signaling between all cell types in the neurovascular unit. Taken together, our proof-of-concept study suggest that NIR light may be an effective conditioning tool to stimulate astrocytic signaling and promote EPC accumulation after stroke.

Keywords: Astrocytes; endothelial progenitor cells; near-infrared light; stroke.

Figure 1:. Near infrared light promotes HMGB1 secretion via an increase of intracellular calcium in rat cortical astrocytes.

a. In vitro rat cortical astrocytes were exposed to NIR light (808 nm, 0, 10, 20, 37 mW/cm² for 2 minutes). Fluo4 AM signal indicated intracellular calcium mobilization after NIR light therapy in astrocytes (n=4-12). b. Immunocytochemistry demonstrated that HMGB1 was translocated from nucleus to cell cytoplasm at 1 hour after NIR irradiation. c. HMGB1 was released from astrocytes at 24 hours after NIR irradiation (n=4-7). d. Intracellular calcium mobilization after NIR stimulation was blocked by EDTA (10 μM) or BAPTA-AM (5 μM) (n=5-8). e. Western blot analysis demonstrated that EDTA (10 μM) or BAPTA-AM (5 μM) significantly suppressed HMGB1 secretion mediated by NIR irradiation (n=5-14). **P<0.01 vs control, ^#P<0.05 vs NIR (4.4). f. NIR did not influence cell viability (n=4). All data are shown as mean±SD.

Figure 2:. Astrocytic HMGB1 increases endothelial progenitor cells (EPC) proliferation in vitro.

a. Schematic experimental design for media transfer experimentss. Conditioned media from empty well (Control), normal astrocytes (ACM), and NIR-stimulated astrocytes (NIR-ACM) were collected at 24 hours after incubation. Each conditioned media was transferred into early EPC cultures (day 5 after seeding) and incubated for 24 hours. b, c. NIR-ACM significantly enhanced early EPC proliferation compared with ACM (n=8). **P<0.01 vs control. d. Loss-of-function study was performed by using siRNA. HMGB1 siRNA successfully suppressed HMGB1 secretion into conditioned media after NIR irradiation. e. NIR-ACM mediated EPC proliferation was significantly suppressed when astrocytic HMGB1 was decrease by siRNA (n=4). *P<0.05. f, g. Conditioned media collected from NIR-stimulated EPC did not increase own proliferation (n=4). All data are shown as mean±SD.

Figure 3:. In vivo transcranial near infrared light increases GFAP expression in the brains of treated C57bl6 mice.

a. Fresh scalp and skull were dissected from the mouse to measure a percentage of NIR-light penetration. b. NIR-light penetration was determined in three independent power irradiation. White circles show the power levels without mouse scalp and skull, and black circles show the powers reduced by fresh scalp and skull isolated from mouse. c. In each irradiation, we confirmed that ~40 % of power was reached into the surface of cerebral cortex. d. Exposure to NIR light (808 nm, 37 mW/cm² for 2 min) was performed with setting illuminator lens 5 mm from the mouse’s shaved scalp. e, f. GFAP positive astrocytes in primary somatosensory cortex and corpus callosum were determined by immunohistochemistry 30 min after NIR irradiation. Immunohistochemistry and western blot showed that compared to control, transcranial laser irradiation increased GFAP signal in both cerebral cortex and corpus callosum (n=3). Scale bar: 100 μm. *P<0.05 vs control. All data are shown as mean±SD.

Figure 4:. In vivo transcranial near infrared light increases EPC accumulation after focal cerebral ischemia in C57bl6 mice.

a. Exposure to NIR light (808 nm, 37 mW/cm² for 2 min) was performed at days 5, 9, and 13 after stroke onset. b. NIR irradiation enhanced HMGB1 expression in GFAP positive reactive astrocyte in per-infarct area at day 14 after stroke. c. Western blot analysis confirmed that NIR upregulated HMGB1 in peri-infarct cortex. d. Immunohistochemistry showed an accumulation of Flk1/CD34 double positive EPCs in somatosensory cortex in peri-infarct region at day 14 after focal ischemia. e. FACS analysis demonstrated that with 3 rounds of NIR irradiation, EPCs were significantly increased in the somatosensory cortex 14 days after stroke onset. f. Transcranial NIR may become a tool to promote endogenous mechanism of crosstalk between reactive astrocytes and EPCs after stroke.