NIR Laser Photobiomodulation Induces Neuroprotection in an In Vitro Model of Cerebral Hypoxia/Ischemia

Abstract

Brain photobiomodulation (PBM) is an innovative treatment for a variety of neurological conditions, including cerebral ischemia. However, the capability of PBM for ischemic stroke needs to be further explored and its mechanisms of action remain currently unclear. The aim of the present research was to identify a treatment protocol capable of inducing neuroprotection and to investigate the molecular mechanisms activated by a dual-wavelength near infrared (NIR) laser source in an organotypic hippocampal slice model of hypoxia/ischemia. Hippocampal slices were exposed to oxygen and glucose deprivation (OGD) for 30 min followed by NIR laser light (fluence 3.71, 7.42, or 14.84 J/cm²; wavelengths 808 nm and 905 nm) delivered immediately or 30 min or 60 min after OGD, in order to establish a therapeutic window. Neuronal injury was assessed by propidium iodide fluorescence 24 h later. Our results show that NIR laser irradiation attenuates OGD neurotoxicity once applied immediately or 30 min after OGD. Western blot analysis of proteins involved in neuroinflammation (iNOS, COX-2, NFkB subunit p65, and Bcl-2) and in glutamatergic-mediated synaptic activity (vGluT1, EAAT2, GluN1, and PSD95) showed that the protein modifications induced by OGD were reverted by NIR laser application. Moreover, CA1 confocal microscopy revealed that the profound morphological changes induced by OGD were reverted by NIR laser radiation. In conclusion, NIR laser radiation attenuates OGD neurotoxicity in organotypic hippocampal slices through attenuation of inflammatory mechanisms. These findings shed light on molecular definition of NIR neuroprotective mechanisms, thus underlining the potential benefit of this technique for the treatment of cerebral ischemia.

Keywords: Glia activation; NIR laser; Neurodegeneration; Organotypic hippocampal slices; Oxygen and glucose deprivation; Photobiomodulation.

Fig. 1

NIR laser treatment induces neuroprotection in rat organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD). A Experimental protocols showing schematic diagrams of NIR laser treatment in organotypic hippocampal slices exposed to 30 min oxygen and glucose deprivation (OGD). B Qualitative analysis representing organotypic hippocampal slice in control condition or exposed to 3.71, 7.42, or 14.84 J/cm² alone or immediately after 30 min OGD and 24 h later incubated with PI for fluorescence detection. C Quantitative analysis of CA1 PI fluorescence expressed as percentage of OGD toxicity. Data are expressed as percentage of OGD-induced PI fluorescence in CA1 region. Bars represent the mean ± SEM of at least four experiments run in quadruplicate. *P < 0.05, **P < 0.01 vs. OGD (ANOVA + Tukey’s w test)

Fig. 2

Therapeutic window of NIR laser treatment in rat organotypic hippocampal slices exposed to OGD. A Qualitative analysis representing organotypic hippocampal slice in control condition or exposed to 7.42 J/cm² immediately or 30 min or 60 min after 30 min OGD and 24 h later incubated with PI for fluorescence detection. B Quantitative analysis of CA1 PI fluorescence expressed as percentage of OGD toxicity. Data are expressed as percentage of OGD-induced PI fluorescence in CA1 region. Bars represent the mean ± SEM of at least four experiments run in quadruplicate. **P < 0.01 vs. OGD (ANOVA + Tukey’s w test)

Fig. 3

Effects of NIR laser treatment after OGD toxicity on proteins involved in neuroinflammation. Experiments were conducted as described in Fig. 1. Hippocampal slices were exposed to 30 min OGD and immediately after to 7.42 J/cm² NIR laser. Twenty-four hours later, the expression of iNOS, COX-2, phospho p65 (NFkB), and Bcl2 was evaluated in total homogenate by Western blot analysis. Data are expressed as percentage of control. Bars represent the mean ± SEM of at least three experiments run in ottuplicate. *P < 0.05, **P < 0.01 vs. CRL, #P < 0.05, ##P < 0.01 vs. OGD (ANOVA + Tukey’s w test)

Fig. 4

Effects of NIR laser treatment after OGD toxicity on proteins involved in excitatory synaptic activity. Experiments were conducted as described in Fig. 1. Hippocampal slices were exposed to 30 min OGD and immediately after to 7.42 J/cm² NIR laser. Twenty-four hours later, the expression of GluN1, PSD95, vGluT1, and EAAT2 was evaluated in total homogenate by Western blot analysis. Data are expressed as percentage of control. Bars represent the mean ± SEM of at least three experiments run in ottuplicate. *P < 0.05, **P < 0.01 vs. CRL, #P < 0.05, ##P < 0.01 vs. OGD (ANOVA + Tukey’s w test)

Fig. 5

Effects of NIR laser treatment on OGD-induced neurotoxicity and glia activation. A–D3 Representative confocal microscopy images showing CA1 immunostaining of NeuN + neurons (red, A1–D1), GFAP + astrocytes (green, A2–D2), and IBA1 + microglia (blue, A3–D3) in CA1 stratum pyramidale of organotypic hippocampal slices and their merge (A–D). Control slices (CTR, A–A3), laser-treated control slices (laser, B–B3), OGD slices (OGD, C–C3), and laser-treated OGD slices (laser + OGD, D–D3). Scale bar: 100 μm. C4 Magnification of the framed area in C1 showing representative examples of NeuN + pyknotic, and HDN neurons in an OGD slice (open arrows). Scale bar: 40 μm. E–G Quantitative analyses of NeuN + HDN neurons (E), GFAP + astrocytes (F), and IBA1 + reactive microglia (G) in CA1 of CTR slices (white column, n = 6), laser slices (light gray column, n = 6), OGD slices (dark gray column, n = 7), and laser + OGD slices (black column, n = 6) (one-way ANOVA, P value = 0.0002; Newman-Keuls post-test: *, **, ***P < 0.05, 0.01, 0.001 vs CTR; #P < 0.05 vs laser; $P < 0.05 vs OGD)

Fig. 6

Graphical conclusion: Representative cartoons describing the effects of NIR laser photobiomodulation on organotypic hippocampal slices exposed to hypoxia/ischemia