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Photobiomodulation on cultured cortical neurons
Chapter 4
Photobiomodulation on cultured cortical neurons Ying-Ying Huang1,2 and Michael R. Hamblin1,2 1
Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, United States, 2Department of Dermatology,
Harvard Medical School, Boston, MA, United States
4.1
Introduction
Photobiomodulation (PBM) has gained considerable interest in recent years as a therapy for the treatment of a variety of diseases and injuries, by eliciting significant biological effects at a cellular level and at a tissular level. PBM is supported by studies of various animal models of different diseases and conditions, and by a large number of clinical trials, many of which were randomized controlled trials. Different conditions for which this therapy has shown positive outcome include pain relief (Peres, 2010; Fulop et al., 2010; Konstantinovic et al., 2010), inflammation (Xavier et al., 2010), and wound healing (Peplow et al., 2010; Lucas et al., 2002; Yasukawa et al., 2007). In the last few years, this therapy has been extended to the treatment of more severe conditions such as stroke, myocardial infarctions, neurodegenerative disorders, and traumatic brain injury (TBI; Hashmi et al., 2010). There are a growing number of reports demonstrating the promising outcome for PBM for diseases related to the central nervous system (CNS). Some of the positive effects shown in neurons are the promotion of axonal growth and nerve regeneration in both rat spinal cord (Byrnes et al., 2005; Wu et al., 2009) and peripheral nerve injuries (Anders et al., 2004) after PBM. The efficacy of PBM in the nervous system has been further established in animal studies showing improved neurological and functional outcomes poststroke (Oron et al., 2006; Detaboada et al., 2006; Lapchak and De Taboada, 2010), postTBI (Oron et al., 2007), and in a mouse model of Alzheimer’s disease (De Taboada et al., 2011). PBM also improved emotional response and memory function of middle-aged CD-1 mice (Michalikova et al., 2008). The results of human clinical studies in patients with long term peripheral nerve injury (Lucas et al., 2002) and ischemic stroke (Lampl et al., 2007; Lapchak, 2010) have also been promising. Recently, a study was performed on two human chronic TBI patients which showed that transcranial LED could improve cognition (Naeser et al., 2011). Although the data for therapeutic usefulness of PBM for neurological disorders is promising, the medical and research community at large is still skeptical about this therapy. The reason for these reservations is the lack of fundamental understanding into the basic mechanistic effects of PBM and how the cellular effects on neurons (that can be observed in cell culture studies) can be translated into improved brain function in vivo. The proposed mechanism of PBM is largely assumed to involve stimulation of the cellular energy metabolism and energy production mediated by mitochondria acting as the primary cellular photoreceptor or target for absorbing the photons. Within mitochondria, cytochrome c oxidase (complex 4 of the respiratory chain) is considered to be the principal chromophore absorbing the incident light; although other cytochromes, porphyrins, and heme proteins could be involved. Light absorption further leads to increased activity of cytochrome c oxidase (Hu et al., 2007), release of nitric oxide (NO) (Karu et al., 2005), and an increase in ATP levels (Pastore et al., 1996; Passarella et al., 1984). Changes in intracellular signaling molecules such as calcium ions, reactive oxygen species (ROS), and redoxsensitive transcription factors like NF-kB are also thought to mediate the effects of light. Previous studies from our laboratory in mouse embryonic fibroblast cells (Chen et al., 2009) have shown that 810 nm laser induced ROS-mediated NF-kB activation.
Photobiomodulation in the Brain. DOI: https://doi.org/10.1016/B978-0-12-815305-5.00004-X © 2019 Elsevier Inc. All rights reserved.
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4.2
PART | I Basic considerations and in vitro
Dose response in cultured cortical neurons
PBM studies have demonstrated that light follows a biphasic response (Huang et al., 2009, 2011), along the lines of the frequently cited “Arndt-Schulz Law” (Lubart et al., 2006; Chow, 2006; Hawkins and Abrahamse, 2006a,b; Sommer et al., 2001). The Arndt-Schulz Law, formulated in 1888, stated that various poisons, if given in very low doses, had a stimulatory effect; small dosages were beneficial and large doses were harmful (Calabrese, 2016). Stated in a generic manner, the law states that small doses stimulate, moderate doses inhibit, and large doses kill. This concept was developed into “hormesis,” which has been extensively reviewed by Edward Calabrese (Agathokleous et al., 2018; Calabrese and Mattson, 2017; Calabrese et al., 2016). Such an axiom fits well for PBM; where, it has been clearly shown that a biphasic dose response exists. An insufficient dose of energy would result in no response (or an insignificant one); if the dose of light is increased past the necessary threshold, then biostimulation takes place. However, if too much energy is applied, then not only does biostimulation decrease, but bioinhibition may also occur if the light dose is increased even further. The biochemical mechanisms responsible for the biphasic dose response of PBM at the cellular level remain unknown, although reactive oxygen species have been implicated. We decided to measure the cellular responses of primary cultured mouse cortical neurons to PBM (810 nm laser) (Sharma et al., 2011). A wide range of fluences spanning three orders of magnitude was tested to increase the chances of detecting the biphasic dose response. Pregnant female C57BL/6 mice (aged 8 10 weeks) were sacrificed at 16 days postconception. The cortical lobes of 6 10 embryonic brains were separated from subcortical structures and dissociated mechanically. Cells were plated at a density of approximately 30,000 cells/well on poly-D-lysine coated cell culture plates or sterile glass coverslips. The plating and maintenance media consisted of Neurobasal plus B27 supplement (NB27). This media formulation inhibits the outgrowth of glia resulting in a neuronal population which is .95% pure (Brewer, 1995). Experiments were performed on days 8 9 after plating the cells. The cells were irradiated in continuous wave mode using an 810 nm laser with a power density of 25 mW/cm2 for different time periods to achieve energy densities of 0.03, 0.3, 3, 10, and 30 J/cm2. Assays were conducted after the end of the longest irradiation period (20 minutes). ROS production was measured using MitoSox Red, a fluorescent indicator of mitochondrial superoxide. Intracellular NO production was measured using DAF-FM (which is essentially nonfluorescent until it is nitrosylated by products of oxidation of NO) resulting in DAF-FM triazole which exhibits about a 160-fold greater fluorescence quantum yield. Intracellular calcium was measured by Fluo-4AM, which exhibits a large fluorescence intensity increase on binding to Ca21. Mitochondrial membrane potential (MMP) was measured by JC1, which is a cationic dye that accumulates in mitochondria. This accumulation is dependent on the membrane potential and indicated by a fluorescence emission shift from green (B525 nm) to red (B590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. ATP was measured by the luminescence-based Cell-Titer Glo Assay. The ROS levels showed an interesting triphasic pattern depicted in Fig. 4.1C. There was a significant initial increase with a fluence as low as 0.03 J/cm2, a further increase at 0.3 J/cm2,and a peak corresponding to a threefold increase over baseline that ROS was seen at 3 J/cm2. When the fluence was further increased, there was a significant decrease (compared to 3 J/cm2) in ROS observed at 10 J/cm2. When the fluence was further increased to 30 J/cm2 there was a second significant increase (compared to 10 J/cm2) in ROS to a level higher (but not significantly) than that observed at 3 J/cm2. PBM gave an increase in intracellular DAF-FM fluorescence in the cortical neurons on irradiating with light as shown in the micrographs in Fig. 4.2A and B after a fluence of 0.3 J/cm2, indicating an increase in nitric oxide levels postirradiation. The NO levels followed the same general triphasic pattern as the ROS levels but the increase was less prominent as compared to the ROS (Fig. 4.2C). There was a significant increase at 0.3 J/cm2 compared to control, a significant decrease at 3 J/cm2 compared to 0.3 J/cm2, and a further increase at 30 J/cm2 compared to 0.3 J/cm2. The whole triphasic pattern appeared to be shifted toward lower fluences (the first peak was at 0.3 J/cm2 instead of 3 J/cm2 and the following trough at 3 J/cm2 instead of 10 J/cm2) compared to the pattern observed with ROS (compare Fig. 4.1C). The changes in MMP were determined by measuring the red to green ratio of the fluorescence of JC1 dye. When the MMP is high, more dye accumulates in the mitochondrial membrane which leads to dye aggregation (J-aggregates) and a red shift in the fluorescence. By contrast, when MMP is low, the dye disaggregates and the fluorescence is green shifted (Reers et al., 1991). The MMP in neurons was significantly increased after 3 J/cm2 of 810-nm light as seen in the micrographs in Fig. 4.3A and B. The dose response was biphasic in nature (Fig. 4.3C). There was a significant increase at 0.3 J/cm2 reaching a peak of twice basal levels at 3 J/cm2. Then there was a decrease at 10 J/cm2, and outright depolarization (significantly lower than control) of MMP was observed at 30 J/cm2.
FIGURE 4.1 Effect of 810-nm PBM on production of mitochondrial ROS in the cultured cortical neurons. (A) Mitosox (red) and nuclear Hoechst (blue) fluorescence in control neurons. (B) Mitosox and nuclear Hoechst fluorescence in neurons treated with 3 J/cm2 810-nm laser. Scale bar is 50 µm. (C) Quantification by fluorescence plate reader of mean Mitosox fluorescence values from 9 wells. Error bars are SD. *P , .05; **P , .01; ÐÐ ***P , .01 versus control. ##P , .01 versus 3 J/cm2. P , .01 versus 10 J/cm2. FIGURE 4.2 Effect of 810-nm laser on production of intracellular NO in the cultured cortical neurons. (A) DAF-DM (green) and nuclear Hoechst (blue) fluorescence in control neurons. (B) DAF-DM and nuclear Hoechst in neurons treated with 0.3 J/cm2 810-nm laser. Scale bar is 50 µm. (C) Quantification by fluorescence plate reader of mean DAF-FM fluorescence values from 9 wells. Error bars are SD. # 2 Ð*P , .05 versus control; P , .05 versus 0.3 J/cm ; P , .05 versus 3 J/cm2.
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FIGURE 4.3 Effect of 810-nm laser on mitochondrial membrane potential in the cultured cortical neurons. (A) JC1 nonaggregated (green), JC1 aggregated (red), and nuclear Hoechst (blue) fluorescence in control neurons. (B) JC1 nonaggregated, JC1 aggregated, and nuclear Hoechst fluorescence in neurons treated with 3 J/cm2 810-nm laser. Scale bar is 50 µm. (C) Quantification by fluorescence plate reader of the mean red/green fluorescence ratio values from 9 wells. Error bars are SD. *P , .05; **P , .05 versus control. ##P , .01; ###P , .001 versus 3 J/cm2.
Neuronal cells treated with PBM showed a significant rise in the intracellular calcium levels as compared to the control as shown in the micrographs in Fig. 4.4A and B captured after 3 J/cm2. The dose response of intracellular calcium was also biphasic as seen in Fig. 4.4C. The increase was significant when it peaked at the fluence of 3 J/cm2. Interestingly, the levels decreased significantly in cells irradiated with 10 and 30 J/cm2. Fig. 4.5 shows the ATP content per mg cell protein in the mouse cortical neurons. ATP content was found to increase on treating the neurons with 810-nm light reaching a significant peak at 3 J/cm2 as compared to the control cells. When the fluence was increased to 10 and 30 J/cm2 the levels of the ATP fell and were equal to that of the control, and in the case of 30 J/cm2 significantly less than the peak at 3 J/cm2.
4.3
Oxidative stress in cultured cortical neurons
Since the previous study, described above (Sharma et al., 2011), had established that 3 J/cm2 of 810 nm laser was the best dose to stimulate cortical neurons, we went on to delve deeper into the question of ROS and oxidative stress, and the cellular response to PBM. We wanted to resolve the apparent paradox, that PBM can increase the levels of ROS in vitro, while it was well-known that PBM could decrease levels of oxidative stress in vivo (Silveira et al., 2011; Lim et al., 2009, 2010; Rubio et al., 2009). We explored the effects of PBM in our model of primary cultured mouse cortical neurons that had been subjected to oxidative stress in vitro by three distinctly different methods. These were: (1) cobalt chloride (CoCl2) which catalyzes the Fenton reaction producing hydroxyl radicals types (Tomaro et al., 1991; Chandel et al., 1998); (2) hydrogen peroxide (H2O2) which is a classical inducer of oxidative stress (Hool and Corry, 2007; Lin et al., 2004); and (3) rotenone which inhibits the mitochondrial respiratory chain at complex I, can cause highly selective degeneration of nigrostriatal neurons, and can reproduce many features of Parkinson’s disease (Freestone et al., 2009; Betarbet et al., 2000). For CoCl2 treatment concentrations of 0.2, 0.5, 1, and 2 mM led to cytotoxicity ranging from 25% to 45% (Fig. 4.6A). PBM treatment with 3 J/cm2 810-nm laser protects neurons from the cytotoxic effects of CoCl2 by reducing the loss of viability to a maximum of 12% cytotoxicity (P , .01). For H2O2, treatment with 10 and 20 µM H2O2 led to cytotoxicity of 67% and 75%, respectively. Laser treatment reduced the neuronal cytotoxicity to 56% (P , .01) and
FIGURE 4.4 Effect of 810-nm laser on intracellular calcium in the cultured cortical neurons. (A) Fluo4 (green) and nuclear Hoechst (blue) fluorescence in control neurons. (B) Fluo4 (green) and nuclear Hoechst (blue) fluorescence in neurons treated with 3 J/cm2 810-nm laser. Scale bar is 50 µm. (C) Quantification by fluorescence plate reader of the mean red/green fluorescence ratio values from 9 wells. Error bars are SD. *P , .05 versus control. # P , .05 versus 3 J/cm2.
FIGURE 4.5 Effect of 810-nm laser on intracellular ATP in the cultured cortical neurons. Quantification by luminescence plate reader of the relative light unit values per mg cell protein from Cell-Titer Glo assay from 9 wells. Error bars are SD. *P , 0.05 versus control. # P , .05 versus 3 J/cm2.
FIGURE 4.6 Cell viability of cortical neurons treated with increasing doses of (A) CoCl2, (B) H2O2, (C) rotenone for 1 h with and without 3 J/cm2 810-nm laser in first 2.5 min. N 5 6 wells per group, error bars are SEM and *P , .05 and **P , .01.
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PART | I Basic considerations and in vitro
FIGURE 4.7 Representative images of cortical neurons labeled with MitoRox (red) for mitochondrial ROS, Mitotracker green for mitochondrial colocalization, and Hoescht (blue) for nuclei (seen in triple overlay). (A) control cortical neurons; (B) cortical neurons with 3 J/cm2 810-nm laser; (C) cortical neurons treated with 500 µM CoCl2; (D) cortical neurons treated with 500 µM CoCl2 and 3 J/cm2 810-nm laser; (E) cortical neurons treated with 20 µM H2O2; (F) cortical neurons treated with 20 µM H2O2 and 3 J/cm2 810-nm laser. (G) cortical neurons treated with 200 nM rotenone; (H) cortical neurons treated with 200 nM rotenone and 3 J/cm2 810-nm laser. Scale bar 5 20 µm. (I) Quantification of fluorescence measurements from the groups shown. N 5 6 fields per group, error bars are SEM and *P , .05 and **P , .01.
58% (P , .05), respectively (Fig. 4.6B). Rotenone treatment (0.1 5 µM) produced a dose-dependent loss of viability reaching cytotoxicity of 94% at 5 µM. Laser treatment protected neurons against rotenone cytotoxicity, however the protection in cell viability was much reduced (only 3% 5% less killing) compared to the other oxidative stress agents. Nevertheless, the protection was statistically significant (P , .05) at 0.2, 2, and 5 µM (Fig. 4.6C). Representative images of the Mitosox and Mitotreacker staining are shown in Fig. 4.7(A H) and the quantification of the fluorescence measurements is shown in Fig. 4.7I. Significant (P , .01) Mitosox red fluorescence indicating mitochondrial production of ROS was seen in control neurons (no oxidative stress) exposed to 3 J/cm2 810 nm laser. This is in agreement with the results we have reported previously (Sharma et al., 2011). An approximate sevenfold rise in mitochondrial ROS was seen in the 500 µM CoCl2 treated group. Mitochondrial ROS production was significantly reduced (P , .05) after exposure to 3 J/cm2 laser. H2O2 (20 µM) also produced an approximate sevenfold rise in mitochondrial ROS, and this was significantly reduced (P , .01) after 3 J/cm2 of 810 nm laser. Rotenone (200 nM) also produced an approximate sevenfold rise in mitochondrial ROS, which was significantly reduced (P , .01) by laser treatment. Representative images of the CellRox fluorescence are shown in Fig. 4.8(A H), and the quantification of the fluorescence measurements is shown in Fig. 4.8I. In contrast to the results that were found with mitochondrial ROS, there was not a significant rise in cytoplasmic ROS when laser was delivered to control neurons without oxidative stress. An approximate fourfold rise in cytoplasmic ROS was seen in the 500 µM CoCl2 treated group. Cytoplasmic ROS production was significantly reduced (P , .001) after exposure to 3 J/cm2 laser. H2O2 (20 µM) produced an approximate 2.5-fold rise in cytoplasmic ROS, and this was significantly reduced (P , .01) after 3 J/cm2 of 810nm laser. Rotenone (200 nM) produced a threefold rise in cytoplasmic ROS, which was significantly reduced (P , .01) by laser treatment. Representative images are shown in Fig. 4.9A H and the quantification of the fluorescence measurements is shown in Fig. 4.9I. In agreement with the results from a previous study (Sharma et al., 2011) we found a small (B30%) but significant (P , .01) rise in MMP when laser was delivered to control neurons without oxidative stress. An approximate threefold reduction in MMP was seen in the 500 µM CoCl2 treated group. MMP was significantly increased (P , .01) after exposure to 3 J/cm2 laser. H2O2 (20 µM) produced an approximate fivefold reduction in MMP, and this was significantly attenuated to control levels (P , .001) after 3 J/cm2 of 810 nm laser. Rotenone (200 nM) produced the largest reduction in MMP and although the increase after laser treatment was relatively minor compared to other oxidative stress agents, it was still significant (P , .01).
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FIGURE 4.8 Representative images of cortical neurons labeled with Cellrox (deep red) for cytoplasmic ROS, and Hoescht (blue) for nuclei. (A) control cortical neurons; (B) cortical neurons with 3 J/cm2 810-nm laser; (C) cortical neurons treated with 500 µM CoCl2; (D) cortical neurons treated with 500 µM CoCl2 and 3 J/cm2 810-nm laser; (E) cortical neurons treated with 20 µM H2O2; (F) cortical neurons treated with 20 µM H2O2 and 3 J/cm2 810-nm laser.;(G) cortical neurons treated with 200 nM rotenone; (H) cortical neurons treated with 200 nM rotenone and 3 J/cm2 810-nm laser. Scale bar 5 20 µm. (I) Quantification of fluorescence measurements from the groups. N 5 6 fields per group, error bars are SEM and *P , .05; **P , .01 and ***P , .001.
4.4
Excitotoxicity in cultured cortical neurons
Excitotoxicity is a pathological process by which neurons are damaged and killed by the excessive stimulation of receptors for the excitatory amino acid neurotransmitter glutamate (Glu) in the CNS. Excitotoxicity may be involved in CNS pathologies such as stroke, TBI, and spinal cord injury, and is also implicated in neurodegenerative diseases such as multiple sclerosis, Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington’s disease. We tested three different protocols to induce excitotoxicity in the neurons in vitro (Huang et al., 2014). These were (1) glutamate 30 µM, (2) N-methyl-D-aspartate (NMDA) 100 µM, and (3) kainic acid (KA) 50 µM; all incubated for 1 hour followed by a wash and exposure to real or sham PBM.
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PART | I Basic considerations and in vitro
FIGURE 4.9 Representative images of cortical neurons labeled with TMRM for mitochondrial membrane potential, and Hoescht (blue) for nuclei. (A) control cortical neurons; (B) cortical neurons with 3 J/cm2 810-nm laser; (C) cortical neurons treated with 500 µM CoCl2; (D) cortical neurons treated with 500 µM CoCl2 and 3 J/cm2 810-nm laser; (E) cortical neurons treated with 20 µM H2O2; (F) cortical neurons treated with 20 µM H2O2 and 3 J/cm2 810-nm laser; (G) cortical neurons treated with 200 nM rotenone; (H) cortical neurons treated with 200 nM rotenone and 3 J/cm2 810-nm laser. Scale bar 5 20 µm. (I) Quantification of fluorescence measurements from the groups. N 5 6 fields per group, error bars are SEM and *P , .05; **P , .01 and ***P , .001.
One hour exposure to either of these excitotoxins followed by 24 h incubation produced reductions in cell viability. Glutamate (30 µM) led to 51% survival, NMDA (100 µM) led to 42% survival, and KA (50 µM) led to 50% survival. PBM treatment produced a modest but significant increase in survival to 65% for glutamate (P , .05), to 71% for NMDA (P , .01), and to 64% for kainate (Fig. 4.10A). Fig. 4.10B shows the ATP content per mg cell protein. ATP content was found to significantly increase (98% more, P , .05) when control neurons were treated with 810nm light. All three excitotoxins significantly reduced the ATP content of the neurons to between 10% and 23% of the control value (P , .001). PBM increased the ATP content of glutamate-treated neurons from 13% to 73% (P , .001), for NMDA-treated neurons from 23% to 80%, and for KA-treated neurons from 10% to 60%. In all three examples of excitotoxicity, the ATP content was at least tripled by PBM.
Photobiomodulation on cultured cortical neurons Chapter | 4
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FIGURE 4.10 Cell viability and ATP. (A) Cell viability measured by Prestoblue and (B) ATP levels measured by Cell-Titer Glo of cortical neurons treated with excitotoxins for 1 h with and without 3 J/cm2 810 nm laser in first 3 min. N 5 6 wells per group, error bars are SEM and *P , .05; **P , .01 and ***P , .001.
FIGURE 4.11 Intracellular calcium measured by Fluo-4. (A) Fluo4 AM (green) fluorescence for calcium and Hoechst (blue) fluorescence for nuclei in excitotoxic cortical neurons with and without 3 J/cm2 810 nm laser. B: Quantification of mean Fluo-4 fluorescence values. N 5 6 fields per group. Error bars are SEM and *P , .05; **P , .01 and ***P , .001. Scale bar 5 20 µm.
Representative confocal microscope images are shown in Fig. 4.11A, and the quantification of the fluorescence measurements is shown in Fig. 4.11B. In agreement with our previously published data (Sharma et al., 2011), a 71% rise in intracellular Ca21 was seen after PBM treatment of control neurons. All three excitotoxins significantly increased the intracellular Ca21 content of the neurons by between 80% and 200% of the dark control value (P , .001). PBM reduced the Ca21 content of glutamate-treated neurons by more than 50% (P , 0.001). PBM produced a smaller decrease in the Ca21 content of NMDA-treated neurons of 25% (P , .01). The decrease in Ca21 content in the kainate-treated neurons produced by PBM was almost as large (43%, P , .01) as that found with glutamate. Representative images are shown in Fig. 4.12A, and the quantification of the fluorescence measurements is shown in Fig. 4.12B. Again in agreement with the results from a previous study (Sharma et al., 2011) we found a 29% rise in MMP (P , .05) when laser was delivered to control neurons without excitotoxicity. The three excitotoxins all produced drops in MMP ranging from 21% to 78% of control values. MMP in glutamate-treated neurons sharply increased by
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PART | I Basic considerations and in vitro
FIGURE 4.12 MMP measured by tetramethylrhodamine. (A) TMRM (red) fluorescence for cytoplasmic ROS and Hoechst (blue) fluorescence for nuclei in excitotoxic cortical neurons with and without PBM. (B) Quantification mean TMRM fluorescence values. N 5 6 fields per group. Error bars are SEM and *P , .05 and **P , .01. Scale bar 5 20 µm.
FIGURE 4.13 ROS production measured by CellRox red. (A) CellRox Red (red) fluorescence for cytoplasmic ROS and Hoechst (blue) fluorescence for nuclei in excitotoxic cortical neurons with and without PBM. (B) Quantification of mean CellRox fluorescence values. N 5 6 fields per group. Error bars are SEM and *P , .05 and **P , .01. Scale bar 5 20 µm.
200% (P , .01) after PBM. The MMP in NMDA-treated neurons increased by 60% (P , .01), and MMP in kainatetreated neurons doubled by PBM (P , .01). Representative images are shown in Fig. 4.13A, and the quantification of the fluorescence measurements is shown in Fig. 4.13B. In agreement with our previous study (Sharma et al., 2011), we found a significant rise (66%, P , .05) in
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FIGURE 4.14 NO measured by DAF2-AM. (A) DAF2-FM (green) fluorescence for NO in excitotoxic cortical neurons with and without PBM. (B) Quantification of mean DAF-FM fluorescence values. N 5 6 fields per group. Error bars are SEM and *P , .05 and **P , .01. Scale bar 5 20 µm.
cytoplasmic ROS when PBM was delivered to control neurons without excitotoxins. The rise in ROS upon addition of excitotoxins ranged from 50% more (NMDA), 110% more (glutamate), and to 200% more (kainate). In this experiment, there was only a significant reduction in ROS (48% less, P , .01) produced by PBM in the case of kainate-treated neurons. The increased ROS produced by glutamate and by NMDA was not significantly affected by PBM. Representative images are shown in Fig. 4.14A, and the quantification of the fluorescence measurements is shown in Fig. 4.14B. There was a rise in NO content of 220% in control neurons treated by PBM (P , .05) in agreement with our previous study (Sharma et al., 2011). All three excitotoxins produced a large increase in NO ranging from 318% for glutamate, 600% for NMDA, and to 677% for kainate (P , .01). PBM reduced the NO content of glutamate-treated neurons by 50% (P , .05). While there was a 20% reduction in NO for NMDA-treated neurons, it was not statistically significant. The NO in kainate-treated neurons was reduced most by 69% (P , .01) by PBM.
Conclusion It is often said that PBM has much bigger effects on diseased, sick, damaged, or stressed cells, and comparatively few effects on normal, healthy, undamaged cells. Our data begin to shed some light on this observation. In normal cells at doses around the optimum value of 3 J/cm2 (which incidentally is a fairly broad peak in the dose response curve), PBM will increase the MMP above baseline, accompanied by increases in ATP, intracellular calcium, accompanied by modest increases in ROS and NO. However, if the dose is increased beyond the optimum value (100 times higher in J/cm2) then the MMP is decreased below baseline, the increases in ATP and calcium are lost, and a second peak of ROS and NO is observed. If the cells are subjected to damaging insults such as oxidative stress or excitotoxicity before PBM, then the picture is somewhat different. Because these toxic treatments lower the MMP (sometimes severely) and also lower the ATP levels and reduce cell viability, the effect of PBM is to raise MMP back toward baseline, and to reverse the decreases in ATP levels and cell viability. The effect of ROS depends on what treatment the cells were subjected to. In the case of oxidative stress, the MMP is lowered, and the mitochondria produce substantial amounts of ROS. Note the ROS measured in the oxidative stress cells are not due per se to the oxidizing agents added, but are produced by the damaged mitochondria. When the mitochondria are restored toward normality, the ROS levels come down. The levels of calcium are highly elevated by excitotoxicity due to the opening of ion channels, and PBM can lower these calcium levels possibly by increasing ATP levels back toward normal. The levels of ROS produced as a consequence of excitotoxicity is probably also due to the lowering of MMP by the high calcium levels.
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Neurons are highly metabolic cells, with very active mitochondria compared to many other cell types within the human body. The beneficial activities of PBM may be more pronounced in neurons than they would be with other cells. Perhaps this explains why the brain does appear to respond particularly well to PBM.
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Laser acupuncture studies should not be included in systematic reviews of phototherapy. Photomed. Laser Surg. 24 (1), 69. De Taboada, L., Yu, J., El-Amouri, S., Gattoni-Celli, S., Richieri, S., McCarthy, T., et al., 2011. Transcranial laser therapy attenuates amyloid-beta peptide neuropathology in amyloid-beta protein precursor transgenic mice. J. Alzheimers Dis. 23 (3), 521 535. Detaboada, L., Ilic, S., Leichliter-Martha, S., Oron, U., Oron, A., Streeter, J., 2006. Transcranial application of low-energy laser irradiation improves neurological deficits in rats following acute stroke. Lasers Surg. Med. 38 (1), 70 73. Freestone, P.S., Chung, K.K., Guatteo, E., Mercuri, N.B., Nicholson, L.F., Lipski, J., 2009. Acute action of rotenone on nigral dopaminergic neurons-involvement of reactive oxygen species and disruption of Ca21 homeostasis. Eur. J. Neurosci. 30 (10), 1849 1859. Fulop, A.M., Dhimmer, S., Deluca, J.R., Johanson, D.D., Lenz, R.V., Patel, K.B., et al., 2010. A meta-analysis of the efficacy of laser phototherapy on pain relief. Clin. J. Pain 26 (8), 729 736. Hashmi, J.T., Huang, Y.Y., Osmani, B.Z., Sharma, S.K., Naeser, M.A., Hamblin, M.R., 2010. Role of low-level laser therapy in neurorehabilitation. PM R 2 (12 Suppl. 2), S292 S305. Hawkins, D., Abrahamse, H., 2006a. Effect of multiple exposures of low-level laser therapy on the cellular responses of wounded human skin fibroblasts. Photomed. Laser Surg. 24 (6), 705 714. Hawkins, D.H., Abrahamse, H., 2006b. The role of laser fluence in cell viability, proliferation, and membrane integrity of wounded human skin fibroblasts following helium-neon laser irradiation. Lasers Surg. Med. 38 (1), 74 83. Hool, L.C., Corry, B., 2007. Redox control of calcium channels: from mechanisms to therapeutic opportunities. Antioxid. Redox Signal. 9 (4), 409 435. Hu, W.P., Wang, J.J., Yu, C.L., Lan, C.C., Chen, G.S., Yu, H.S., 2007. Helium-neon laser irradiation stimulates cell proliferation through photostimulatory effects in mitochondria. J. Invest. Dermatol. 127 (8), 2048 2057. Huang, Y.Y., Chen, A.C., Carroll, J.D., Hamblin, M.R., 2009. Biphasic dose response in low level light therapy. Dose Response 7 (4), 358 383. Huang, Y.Y., Sharma, S.K., Carroll, J.D., Hamblin, M.R., 2011. Biphasic dose response in low level light therapy an update. Dose Response 9 (4), 602 618. Huang, Y.Y., Nagata, K., Tedford, C.E., Hamblin, M.R., 2014. Low-level laser therapy (810 nm) protects primary cortical neurons against excitotoxicity in vitro. J. Biophotonics 7 (8), 656 664. Karu, T.I., Pyatibrat, L.V., Afanasyeva, N.I., 2005. Cellular effects of low power laser therapy can be mediated by nitric oxide. Lasers Surg. Med. 36 (4), 307 314. Konstantinovic, L.M., Cutovic, M.R., Milovanovic, A.N., Jovic, S.J., Dragin, A.S., Letic, M., et al., 2010. Low-level laser therapy for acute neck pain with radiculopathy: a double-blind placebo-controlled randomized study. Pain Med. 11 (8), 1169 1178. Lampl, Y., Zivin, J.A., Fisher, M., Lew, R., Welin, L., Dahlof, B., et al., 2007. Infrared laser therapy for ischemic stroke: a new treatment strategy: results of the NeuroThera Effectiveness and Safety Trial-1 (NEST-1). Stroke 38 (6), 1843 1849. Lapchak, P.A., 2010. Taking a light approach to treating acute ischemic stroke patients: transcranial near-infrared laser therapy translational science. Ann. Med. 42 (8), 576 586. Lapchak, P.A., De Taboada, L., 2010. Transcranial near infrared laser treatment (NILT) increases cortical adenosine-5’-triphosphate (ATP) content following embolic strokes in rabbits. Brain Res. 1306, 100 105.
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Lim, J., Ali, Z.M., Sanders, R.A., Snyder, A.C., Eells, J.T., Henshel, D.S., et al., 2009. Effects of low-level light therapy on hepatic antioxidant defense in acute and chronic diabetic rats. J. Biochem. Mol. Toxicol. 23 (1), 1 8. Lim, J., Sanders, R.A., Snyder, A.C., Eells, J.T., Henshel, D.S., Watkins 3rd, J.B., 2010. Effects of low-level light therapy on streptozotocin-induced diabetic kidney. J. Photochem. Photobiol. B 99 (2), 105 110. Lin, H.J., Wang, X., Shaffer, K.M., Sasaki, C.Y., Ma, W., 2004. Characterization of H2O2-induced acute apoptosis in cultured neural stem/progenitor cells. FEBS Lett. 570 (1-3), 102 106. Lubart, R., Lavi, R., Friedmann, H., Rochkind, S., 2006. Photochemistry and photobiology of light absorption by living cells. Photomed. Laser Surg. 24 (2), 179 185. Lucas, C., Criens-Poublon, L.J., Cockrell, C.T., de Haan, R.J., 2002. Wound healing in cell studies and animal model experiments by Low Level Laser Therapy; were clinical studies justified? a systematic review. Lasers Med. Sci. 17 (2), 110 134. Michalikova, S., Ennaceur, A., van Rensburg, R., Chazot, P.L., 2008. Emotional responses and memory performance of middle-aged CD1 mice in a 3D maze: effects of low infrared light. Neurobiol. Learn. Mem. 89 (4), 480 488. Naeser, M.A., Saltmarche, A., Krengel, M.H., Hamblin, M.R., Knight, J.A., 2011. Improved cognitive function after transcranial, light-emitting diode treatments in chronic, traumatic brain injury: two case reports. Photomed. Laser Surg. 29, 351 358. Oron, A., Oron, U., Chen, J., Eilam, A., Zhang, C., Sadeh, M., et al., 2006. Low-level laser therapy applied transcranially to rats after induction of stroke significantly reduces long-term neurological deficits. Stroke 37 (10), 2620 2624. Oron, A., Oron, U., Streeter, J., de Taboada, L., Alexandrovich, A., Trembovler, V., et al., 2007. Low-level laser therapy applied transcranially to mice following traumatic brain injury significantly reduces long-term neurological deficits. J. Neurotrauma 24 (4), 651 656. Passarella, S., Casamassima, E., Molinari, S., Pastore, D., Quagliariello, E., Catalano, I.M., et al., 1984. Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by helium-neon laser. FEBS Lett. 175 (1), 95 99. Pastore, D., Di Martino, C., Bosco, G., Passarella, S., 1996. Stimulation of ATP synthesis via oxidative phosphorylation in wheat mitochondria irradiated with helium-neon laser. Biochem. Mol. Biol. Int. 39 (1), 149 157. Peplow, P.V., Chung, T.Y., Baxter, G.D., 2010. Laser photobiomodulation of wound healing: a review of experimental studies in mouse and rat animal models. Photomed. Laser Surg. 28 (3), 291 325. Peres, M.F., 2010. Low-level laser therapy for neck pain. Cephalalgia 30 (11), 1408. Reers, M., Smith, T.W., Chen, L.B., 1991. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30 (18), 4480 4486. Rubio, C.R., Simes, J.C., Moya, M., Soriano, F., Palma, J.A., Campana, V., 2009. Inflammatory and oxidative stress markers in experimental crystalopathy: their modification by photostimulation. Photomed. Laser Surg. 27 (1), 79 84. Sharma, S.K., Kharkwal, G.B., Sajo, M., Huang, Y.Y., De Taboada, L., McCarthy, T., et al., 2011. Dose response effects of 810 nm laser light on mouse primary cortical neurons. Lasers Surg. Med. 43 (8), 851 859. Silveira, P.C., Silva, L.A., Freitas, T.P., Latini, A., Pinho, R.A., 2011. Effects of low-power laser irradiation (LPLI) at different wavelengths and doses on oxidative stress and fibrogenesis parameters in an animal model of wound healing. Lasers Med. Sci. 26 (1), 125 131. Sommer, A.P., Pinheiro, A.L., Mester, A.R., Franke, R.P., Whelan, H.T., 2001. Biostimulatory windows in low-intensity laser activation: lasers, scanners, and NASA’s light-emitting diode array system. J. Clin. Laser Med. Surg. 19 (1), 29 33. Tomaro, M.L., Frydman, J., Frydman, R.B., 1991. Heme oxygenase induction by CoCl2, Co-protoporphyrin IX, phenylhydrazine, and diamide: evidence for oxidative stress involvement. Arch. Biochem. Biophys. 286 (2), 610 617. Wu, X., Dmitriev, A.E., Cardoso, M.J., Viers-Costello, A.G., Borke, R.C., Streeter, J., et al., 2009. 810 nm wavelength light: an effective therapy for transected or contused rat spinal cord. Lasers Surg. Med. 41 (1), 36 41. Xavier, M., David, D.R., de Souza, R.A., Arrieiro, A.N., Miranda, H., Santana, E.T., et al., 2010. Anti-inflammatory effects of low-level light emitting diode therapy on Achilles tendinitis in rats. Lasers Surg. Med. 42 (6), 553 558. Yasukawa, A., Hrui, H., Koyama, Y., Nagai, M., Takakuda, K., 2007. The effect of low reactive-level laser therapy (LLLT) with helium-neon laser on operative wound healing in a rat model. J. Vet. Med. Sci. 69 (8), 799 806.
Photobiomodulation on cultured cortical neurons Ying-Ying Huang1,2 and Michael R. Hamblin1,2 1
Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, United States, 2Department of Dermatology,
Harvard Medical School, Boston, MA, United States
4.1
Introduction
Photobiomodulation (PBM) has gained considerable interest in recent years as a therapy for the treatment of a variety of diseases and injuries, by eliciting significant biological effects at a cellular level and at a tissular level. PBM is supported by studies of various animal models of different diseases and conditions, and by a large number of clinical trials, many of which were randomized controlled trials. Different conditions for which this therapy has shown positive outcome include pain relief (Peres, 2010; Fulop et al., 2010; Konstantinovic et al., 2010), inflammation (Xavier et al., 2010), and wound healing (Peplow et al., 2010; Lucas et al., 2002; Yasukawa et al., 2007). In the last few years, this therapy has been extended to the treatment of more severe conditions such as stroke, myocardial infarctions, neurodegenerative disorders, and traumatic brain injury (TBI; Hashmi et al., 2010). There are a growing number of reports demonstrating the promising outcome for PBM for diseases related to the central nervous system (CNS). Some of the positive effects shown in neurons are the promotion of axonal growth and nerve regeneration in both rat spinal cord (Byrnes et al., 2005; Wu et al., 2009) and peripheral nerve injuries (Anders et al., 2004) after PBM. The efficacy of PBM in the nervous system has been further established in animal studies showing improved neurological and functional outcomes poststroke (Oron et al., 2006; Detaboada et al., 2006; Lapchak and De Taboada, 2010), postTBI (Oron et al., 2007), and in a mouse model of Alzheimer’s disease (De Taboada et al., 2011). PBM also improved emotional response and memory function of middle-aged CD-1 mice (Michalikova et al., 2008). The results of human clinical studies in patients with long term peripheral nerve injury (Lucas et al., 2002) and ischemic stroke (Lampl et al., 2007; Lapchak, 2010) have also been promising. Recently, a study was performed on two human chronic TBI patients which showed that transcranial LED could improve cognition (Naeser et al., 2011). Although the data for therapeutic usefulness of PBM for neurological disorders is promising, the medical and research community at large is still skeptical about this therapy. The reason for these reservations is the lack of fundamental understanding into the basic mechanistic effects of PBM and how the cellular effects on neurons (that can be observed in cell culture studies) can be translated into improved brain function in vivo. The proposed mechanism of PBM is largely assumed to involve stimulation of the cellular energy metabolism and energy production mediated by mitochondria acting as the primary cellular photoreceptor or target for absorbing the photons. Within mitochondria, cytochrome c oxidase (complex 4 of the respiratory chain) is considered to be the principal chromophore absorbing the incident light; although other cytochromes, porphyrins, and heme proteins could be involved. Light absorption further leads to increased activity of cytochrome c oxidase (Hu et al., 2007), release of nitric oxide (NO) (Karu et al., 2005), and an increase in ATP levels (Pastore et al., 1996; Passarella et al., 1984). Changes in intracellular signaling molecules such as calcium ions, reactive oxygen species (ROS), and redoxsensitive transcription factors like NF-kB are also thought to mediate the effects of light. Previous studies from our laboratory in mouse embryonic fibroblast cells (Chen et al., 2009) have shown that 810 nm laser induced ROS-mediated NF-kB activation.
Photobiomodulation in the Brain. DOI: https://doi.org/10.1016/B978-0-12-815305-5.00004-X © 2019 Elsevier Inc. All rights reserved.
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4.2
PART | I Basic considerations and in vitro
Dose response in cultured cortical neurons
PBM studies have demonstrated that light follows a biphasic response (Huang et al., 2009, 2011), along the lines of the frequently cited “Arndt-Schulz Law” (Lubart et al., 2006; Chow, 2006; Hawkins and Abrahamse, 2006a,b; Sommer et al., 2001). The Arndt-Schulz Law, formulated in 1888, stated that various poisons, if given in very low doses, had a stimulatory effect; small dosages were beneficial and large doses were harmful (Calabrese, 2016). Stated in a generic manner, the law states that small doses stimulate, moderate doses inhibit, and large doses kill. This concept was developed into “hormesis,” which has been extensively reviewed by Edward Calabrese (Agathokleous et al., 2018; Calabrese and Mattson, 2017; Calabrese et al., 2016). Such an axiom fits well for PBM; where, it has been clearly shown that a biphasic dose response exists. An insufficient dose of energy would result in no response (or an insignificant one); if the dose of light is increased past the necessary threshold, then biostimulation takes place. However, if too much energy is applied, then not only does biostimulation decrease, but bioinhibition may also occur if the light dose is increased even further. The biochemical mechanisms responsible for the biphasic dose response of PBM at the cellular level remain unknown, although reactive oxygen species have been implicated. We decided to measure the cellular responses of primary cultured mouse cortical neurons to PBM (810 nm laser) (Sharma et al., 2011). A wide range of fluences spanning three orders of magnitude was tested to increase the chances of detecting the biphasic dose response. Pregnant female C57BL/6 mice (aged 8 10 weeks) were sacrificed at 16 days postconception. The cortical lobes of 6 10 embryonic brains were separated from subcortical structures and dissociated mechanically. Cells were plated at a density of approximately 30,000 cells/well on poly-D-lysine coated cell culture plates or sterile glass coverslips. The plating and maintenance media consisted of Neurobasal plus B27 supplement (NB27). This media formulation inhibits the outgrowth of glia resulting in a neuronal population which is .95% pure (Brewer, 1995). Experiments were performed on days 8 9 after plating the cells. The cells were irradiated in continuous wave mode using an 810 nm laser with a power density of 25 mW/cm2 for different time periods to achieve energy densities of 0.03, 0.3, 3, 10, and 30 J/cm2. Assays were conducted after the end of the longest irradiation period (20 minutes). ROS production was measured using MitoSox Red, a fluorescent indicator of mitochondrial superoxide. Intracellular NO production was measured using DAF-FM (which is essentially nonfluorescent until it is nitrosylated by products of oxidation of NO) resulting in DAF-FM triazole which exhibits about a 160-fold greater fluorescence quantum yield. Intracellular calcium was measured by Fluo-4AM, which exhibits a large fluorescence intensity increase on binding to Ca21. Mitochondrial membrane potential (MMP) was measured by JC1, which is a cationic dye that accumulates in mitochondria. This accumulation is dependent on the membrane potential and indicated by a fluorescence emission shift from green (B525 nm) to red (B590 nm). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. ATP was measured by the luminescence-based Cell-Titer Glo Assay. The ROS levels showed an interesting triphasic pattern depicted in Fig. 4.1C. There was a significant initial increase with a fluence as low as 0.03 J/cm2, a further increase at 0.3 J/cm2,and a peak corresponding to a threefold increase over baseline that ROS was seen at 3 J/cm2. When the fluence was further increased, there was a significant decrease (compared to 3 J/cm2) in ROS observed at 10 J/cm2. When the fluence was further increased to 30 J/cm2 there was a second significant increase (compared to 10 J/cm2) in ROS to a level higher (but not significantly) than that observed at 3 J/cm2. PBM gave an increase in intracellular DAF-FM fluorescence in the cortical neurons on irradiating with light as shown in the micrographs in Fig. 4.2A and B after a fluence of 0.3 J/cm2, indicating an increase in nitric oxide levels postirradiation. The NO levels followed the same general triphasic pattern as the ROS levels but the increase was less prominent as compared to the ROS (Fig. 4.2C). There was a significant increase at 0.3 J/cm2 compared to control, a significant decrease at 3 J/cm2 compared to 0.3 J/cm2, and a further increase at 30 J/cm2 compared to 0.3 J/cm2. The whole triphasic pattern appeared to be shifted toward lower fluences (the first peak was at 0.3 J/cm2 instead of 3 J/cm2 and the following trough at 3 J/cm2 instead of 10 J/cm2) compared to the pattern observed with ROS (compare Fig. 4.1C). The changes in MMP were determined by measuring the red to green ratio of the fluorescence of JC1 dye. When the MMP is high, more dye accumulates in the mitochondrial membrane which leads to dye aggregation (J-aggregates) and a red shift in the fluorescence. By contrast, when MMP is low, the dye disaggregates and the fluorescence is green shifted (Reers et al., 1991). The MMP in neurons was significantly increased after 3 J/cm2 of 810-nm light as seen in the micrographs in Fig. 4.3A and B. The dose response was biphasic in nature (Fig. 4.3C). There was a significant increase at 0.3 J/cm2 reaching a peak of twice basal levels at 3 J/cm2. Then there was a decrease at 10 J/cm2, and outright depolarization (significantly lower than control) of MMP was observed at 30 J/cm2.
FIGURE 4.1 Effect of 810-nm PBM on production of mitochondrial ROS in the cultured cortical neurons. (A) Mitosox (red) and nuclear Hoechst (blue) fluorescence in control neurons. (B) Mitosox and nuclear Hoechst fluorescence in neurons treated with 3 J/cm2 810-nm laser. Scale bar is 50 µm. (C) Quantification by fluorescence plate reader of mean Mitosox fluorescence values from 9 wells. Error bars are SD. *P , .05; **P , .01; ÐÐ ***P , .01 versus control. ##P , .01 versus 3 J/cm2. P , .01 versus 10 J/cm2. FIGURE 4.2 Effect of 810-nm laser on production of intracellular NO in the cultured cortical neurons. (A) DAF-DM (green) and nuclear Hoechst (blue) fluorescence in control neurons. (B) DAF-DM and nuclear Hoechst in neurons treated with 0.3 J/cm2 810-nm laser. Scale bar is 50 µm. (C) Quantification by fluorescence plate reader of mean DAF-FM fluorescence values from 9 wells. Error bars are SD. # 2 Ð*P , .05 versus control; P , .05 versus 0.3 J/cm ; P , .05 versus 3 J/cm2.
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PART | I Basic considerations and in vitro
FIGURE 4.3 Effect of 810-nm laser on mitochondrial membrane potential in the cultured cortical neurons. (A) JC1 nonaggregated (green), JC1 aggregated (red), and nuclear Hoechst (blue) fluorescence in control neurons. (B) JC1 nonaggregated, JC1 aggregated, and nuclear Hoechst fluorescence in neurons treated with 3 J/cm2 810-nm laser. Scale bar is 50 µm. (C) Quantification by fluorescence plate reader of the mean red/green fluorescence ratio values from 9 wells. Error bars are SD. *P , .05; **P , .05 versus control. ##P , .01; ###P , .001 versus 3 J/cm2.
Neuronal cells treated with PBM showed a significant rise in the intracellular calcium levels as compared to the control as shown in the micrographs in Fig. 4.4A and B captured after 3 J/cm2. The dose response of intracellular calcium was also biphasic as seen in Fig. 4.4C. The increase was significant when it peaked at the fluence of 3 J/cm2. Interestingly, the levels decreased significantly in cells irradiated with 10 and 30 J/cm2. Fig. 4.5 shows the ATP content per mg cell protein in the mouse cortical neurons. ATP content was found to increase on treating the neurons with 810-nm light reaching a significant peak at 3 J/cm2 as compared to the control cells. When the fluence was increased to 10 and 30 J/cm2 the levels of the ATP fell and were equal to that of the control, and in the case of 30 J/cm2 significantly less than the peak at 3 J/cm2.
4.3
Oxidative stress in cultured cortical neurons
Since the previous study, described above (Sharma et al., 2011), had established that 3 J/cm2 of 810 nm laser was the best dose to stimulate cortical neurons, we went on to delve deeper into the question of ROS and oxidative stress, and the cellular response to PBM. We wanted to resolve the apparent paradox, that PBM can increase the levels of ROS in vitro, while it was well-known that PBM could decrease levels of oxidative stress in vivo (Silveira et al., 2011; Lim et al., 2009, 2010; Rubio et al., 2009). We explored the effects of PBM in our model of primary cultured mouse cortical neurons that had been subjected to oxidative stress in vitro by three distinctly different methods. These were: (1) cobalt chloride (CoCl2) which catalyzes the Fenton reaction producing hydroxyl radicals types (Tomaro et al., 1991; Chandel et al., 1998); (2) hydrogen peroxide (H2O2) which is a classical inducer of oxidative stress (Hool and Corry, 2007; Lin et al., 2004); and (3) rotenone which inhibits the mitochondrial respiratory chain at complex I, can cause highly selective degeneration of nigrostriatal neurons, and can reproduce many features of Parkinson’s disease (Freestone et al., 2009; Betarbet et al., 2000). For CoCl2 treatment concentrations of 0.2, 0.5, 1, and 2 mM led to cytotoxicity ranging from 25% to 45% (Fig. 4.6A). PBM treatment with 3 J/cm2 810-nm laser protects neurons from the cytotoxic effects of CoCl2 by reducing the loss of viability to a maximum of 12% cytotoxicity (P , .01). For H2O2, treatment with 10 and 20 µM H2O2 led to cytotoxicity of 67% and 75%, respectively. Laser treatment reduced the neuronal cytotoxicity to 56% (P , .01) and
FIGURE 4.4 Effect of 810-nm laser on intracellular calcium in the cultured cortical neurons. (A) Fluo4 (green) and nuclear Hoechst (blue) fluorescence in control neurons. (B) Fluo4 (green) and nuclear Hoechst (blue) fluorescence in neurons treated with 3 J/cm2 810-nm laser. Scale bar is 50 µm. (C) Quantification by fluorescence plate reader of the mean red/green fluorescence ratio values from 9 wells. Error bars are SD. *P , .05 versus control. # P , .05 versus 3 J/cm2.
FIGURE 4.5 Effect of 810-nm laser on intracellular ATP in the cultured cortical neurons. Quantification by luminescence plate reader of the relative light unit values per mg cell protein from Cell-Titer Glo assay from 9 wells. Error bars are SD. *P , 0.05 versus control. # P , .05 versus 3 J/cm2.
FIGURE 4.6 Cell viability of cortical neurons treated with increasing doses of (A) CoCl2, (B) H2O2, (C) rotenone for 1 h with and without 3 J/cm2 810-nm laser in first 2.5 min. N 5 6 wells per group, error bars are SEM and *P , .05 and **P , .01.
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PART | I Basic considerations and in vitro
FIGURE 4.7 Representative images of cortical neurons labeled with MitoRox (red) for mitochondrial ROS, Mitotracker green for mitochondrial colocalization, and Hoescht (blue) for nuclei (seen in triple overlay). (A) control cortical neurons; (B) cortical neurons with 3 J/cm2 810-nm laser; (C) cortical neurons treated with 500 µM CoCl2; (D) cortical neurons treated with 500 µM CoCl2 and 3 J/cm2 810-nm laser; (E) cortical neurons treated with 20 µM H2O2; (F) cortical neurons treated with 20 µM H2O2 and 3 J/cm2 810-nm laser. (G) cortical neurons treated with 200 nM rotenone; (H) cortical neurons treated with 200 nM rotenone and 3 J/cm2 810-nm laser. Scale bar 5 20 µm. (I) Quantification of fluorescence measurements from the groups shown. N 5 6 fields per group, error bars are SEM and *P , .05 and **P , .01.
58% (P , .05), respectively (Fig. 4.6B). Rotenone treatment (0.1 5 µM) produced a dose-dependent loss of viability reaching cytotoxicity of 94% at 5 µM. Laser treatment protected neurons against rotenone cytotoxicity, however the protection in cell viability was much reduced (only 3% 5% less killing) compared to the other oxidative stress agents. Nevertheless, the protection was statistically significant (P , .05) at 0.2, 2, and 5 µM (Fig. 4.6C). Representative images of the Mitosox and Mitotreacker staining are shown in Fig. 4.7(A H) and the quantification of the fluorescence measurements is shown in Fig. 4.7I. Significant (P , .01) Mitosox red fluorescence indicating mitochondrial production of ROS was seen in control neurons (no oxidative stress) exposed to 3 J/cm2 810 nm laser. This is in agreement with the results we have reported previously (Sharma et al., 2011). An approximate sevenfold rise in mitochondrial ROS was seen in the 500 µM CoCl2 treated group. Mitochondrial ROS production was significantly reduced (P , .05) after exposure to 3 J/cm2 laser. H2O2 (20 µM) also produced an approximate sevenfold rise in mitochondrial ROS, and this was significantly reduced (P , .01) after 3 J/cm2 of 810 nm laser. Rotenone (200 nM) also produced an approximate sevenfold rise in mitochondrial ROS, which was significantly reduced (P , .01) by laser treatment. Representative images of the CellRox fluorescence are shown in Fig. 4.8(A H), and the quantification of the fluorescence measurements is shown in Fig. 4.8I. In contrast to the results that were found with mitochondrial ROS, there was not a significant rise in cytoplasmic ROS when laser was delivered to control neurons without oxidative stress. An approximate fourfold rise in cytoplasmic ROS was seen in the 500 µM CoCl2 treated group. Cytoplasmic ROS production was significantly reduced (P , .001) after exposure to 3 J/cm2 laser. H2O2 (20 µM) produced an approximate 2.5-fold rise in cytoplasmic ROS, and this was significantly reduced (P , .01) after 3 J/cm2 of 810nm laser. Rotenone (200 nM) produced a threefold rise in cytoplasmic ROS, which was significantly reduced (P , .01) by laser treatment. Representative images are shown in Fig. 4.9A H and the quantification of the fluorescence measurements is shown in Fig. 4.9I. In agreement with the results from a previous study (Sharma et al., 2011) we found a small (B30%) but significant (P , .01) rise in MMP when laser was delivered to control neurons without oxidative stress. An approximate threefold reduction in MMP was seen in the 500 µM CoCl2 treated group. MMP was significantly increased (P , .01) after exposure to 3 J/cm2 laser. H2O2 (20 µM) produced an approximate fivefold reduction in MMP, and this was significantly attenuated to control levels (P , .001) after 3 J/cm2 of 810 nm laser. Rotenone (200 nM) produced the largest reduction in MMP and although the increase after laser treatment was relatively minor compared to other oxidative stress agents, it was still significant (P , .01).
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FIGURE 4.8 Representative images of cortical neurons labeled with Cellrox (deep red) for cytoplasmic ROS, and Hoescht (blue) for nuclei. (A) control cortical neurons; (B) cortical neurons with 3 J/cm2 810-nm laser; (C) cortical neurons treated with 500 µM CoCl2; (D) cortical neurons treated with 500 µM CoCl2 and 3 J/cm2 810-nm laser; (E) cortical neurons treated with 20 µM H2O2; (F) cortical neurons treated with 20 µM H2O2 and 3 J/cm2 810-nm laser.;(G) cortical neurons treated with 200 nM rotenone; (H) cortical neurons treated with 200 nM rotenone and 3 J/cm2 810-nm laser. Scale bar 5 20 µm. (I) Quantification of fluorescence measurements from the groups. N 5 6 fields per group, error bars are SEM and *P , .05; **P , .01 and ***P , .001.
4.4
Excitotoxicity in cultured cortical neurons
Excitotoxicity is a pathological process by which neurons are damaged and killed by the excessive stimulation of receptors for the excitatory amino acid neurotransmitter glutamate (Glu) in the CNS. Excitotoxicity may be involved in CNS pathologies such as stroke, TBI, and spinal cord injury, and is also implicated in neurodegenerative diseases such as multiple sclerosis, Alzheimer’s disease, amyotrophic lateral sclerosis, Parkinson’s disease, and Huntington’s disease. We tested three different protocols to induce excitotoxicity in the neurons in vitro (Huang et al., 2014). These were (1) glutamate 30 µM, (2) N-methyl-D-aspartate (NMDA) 100 µM, and (3) kainic acid (KA) 50 µM; all incubated for 1 hour followed by a wash and exposure to real or sham PBM.
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PART | I Basic considerations and in vitro
FIGURE 4.9 Representative images of cortical neurons labeled with TMRM for mitochondrial membrane potential, and Hoescht (blue) for nuclei. (A) control cortical neurons; (B) cortical neurons with 3 J/cm2 810-nm laser; (C) cortical neurons treated with 500 µM CoCl2; (D) cortical neurons treated with 500 µM CoCl2 and 3 J/cm2 810-nm laser; (E) cortical neurons treated with 20 µM H2O2; (F) cortical neurons treated with 20 µM H2O2 and 3 J/cm2 810-nm laser; (G) cortical neurons treated with 200 nM rotenone; (H) cortical neurons treated with 200 nM rotenone and 3 J/cm2 810-nm laser. Scale bar 5 20 µm. (I) Quantification of fluorescence measurements from the groups. N 5 6 fields per group, error bars are SEM and *P , .05; **P , .01 and ***P , .001.
One hour exposure to either of these excitotoxins followed by 24 h incubation produced reductions in cell viability. Glutamate (30 µM) led to 51% survival, NMDA (100 µM) led to 42% survival, and KA (50 µM) led to 50% survival. PBM treatment produced a modest but significant increase in survival to 65% for glutamate (P , .05), to 71% for NMDA (P , .01), and to 64% for kainate (Fig. 4.10A). Fig. 4.10B shows the ATP content per mg cell protein. ATP content was found to significantly increase (98% more, P , .05) when control neurons were treated with 810nm light. All three excitotoxins significantly reduced the ATP content of the neurons to between 10% and 23% of the control value (P , .001). PBM increased the ATP content of glutamate-treated neurons from 13% to 73% (P , .001), for NMDA-treated neurons from 23% to 80%, and for KA-treated neurons from 10% to 60%. In all three examples of excitotoxicity, the ATP content was at least tripled by PBM.
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FIGURE 4.10 Cell viability and ATP. (A) Cell viability measured by Prestoblue and (B) ATP levels measured by Cell-Titer Glo of cortical neurons treated with excitotoxins for 1 h with and without 3 J/cm2 810 nm laser in first 3 min. N 5 6 wells per group, error bars are SEM and *P , .05; **P , .01 and ***P , .001.
FIGURE 4.11 Intracellular calcium measured by Fluo-4. (A) Fluo4 AM (green) fluorescence for calcium and Hoechst (blue) fluorescence for nuclei in excitotoxic cortical neurons with and without 3 J/cm2 810 nm laser. B: Quantification of mean Fluo-4 fluorescence values. N 5 6 fields per group. Error bars are SEM and *P , .05; **P , .01 and ***P , .001. Scale bar 5 20 µm.
Representative confocal microscope images are shown in Fig. 4.11A, and the quantification of the fluorescence measurements is shown in Fig. 4.11B. In agreement with our previously published data (Sharma et al., 2011), a 71% rise in intracellular Ca21 was seen after PBM treatment of control neurons. All three excitotoxins significantly increased the intracellular Ca21 content of the neurons by between 80% and 200% of the dark control value (P , .001). PBM reduced the Ca21 content of glutamate-treated neurons by more than 50% (P , 0.001). PBM produced a smaller decrease in the Ca21 content of NMDA-treated neurons of 25% (P , .01). The decrease in Ca21 content in the kainate-treated neurons produced by PBM was almost as large (43%, P , .01) as that found with glutamate. Representative images are shown in Fig. 4.12A, and the quantification of the fluorescence measurements is shown in Fig. 4.12B. Again in agreement with the results from a previous study (Sharma et al., 2011) we found a 29% rise in MMP (P , .05) when laser was delivered to control neurons without excitotoxicity. The three excitotoxins all produced drops in MMP ranging from 21% to 78% of control values. MMP in glutamate-treated neurons sharply increased by
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PART | I Basic considerations and in vitro
FIGURE 4.12 MMP measured by tetramethylrhodamine. (A) TMRM (red) fluorescence for cytoplasmic ROS and Hoechst (blue) fluorescence for nuclei in excitotoxic cortical neurons with and without PBM. (B) Quantification mean TMRM fluorescence values. N 5 6 fields per group. Error bars are SEM and *P , .05 and **P , .01. Scale bar 5 20 µm.
FIGURE 4.13 ROS production measured by CellRox red. (A) CellRox Red (red) fluorescence for cytoplasmic ROS and Hoechst (blue) fluorescence for nuclei in excitotoxic cortical neurons with and without PBM. (B) Quantification of mean CellRox fluorescence values. N 5 6 fields per group. Error bars are SEM and *P , .05 and **P , .01. Scale bar 5 20 µm.
200% (P , .01) after PBM. The MMP in NMDA-treated neurons increased by 60% (P , .01), and MMP in kainatetreated neurons doubled by PBM (P , .01). Representative images are shown in Fig. 4.13A, and the quantification of the fluorescence measurements is shown in Fig. 4.13B. In agreement with our previous study (Sharma et al., 2011), we found a significant rise (66%, P , .05) in
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FIGURE 4.14 NO measured by DAF2-AM. (A) DAF2-FM (green) fluorescence for NO in excitotoxic cortical neurons with and without PBM. (B) Quantification of mean DAF-FM fluorescence values. N 5 6 fields per group. Error bars are SEM and *P , .05 and **P , .01. Scale bar 5 20 µm.
cytoplasmic ROS when PBM was delivered to control neurons without excitotoxins. The rise in ROS upon addition of excitotoxins ranged from 50% more (NMDA), 110% more (glutamate), and to 200% more (kainate). In this experiment, there was only a significant reduction in ROS (48% less, P , .01) produced by PBM in the case of kainate-treated neurons. The increased ROS produced by glutamate and by NMDA was not significantly affected by PBM. Representative images are shown in Fig. 4.14A, and the quantification of the fluorescence measurements is shown in Fig. 4.14B. There was a rise in NO content of 220% in control neurons treated by PBM (P , .05) in agreement with our previous study (Sharma et al., 2011). All three excitotoxins produced a large increase in NO ranging from 318% for glutamate, 600% for NMDA, and to 677% for kainate (P , .01). PBM reduced the NO content of glutamate-treated neurons by 50% (P , .05). While there was a 20% reduction in NO for NMDA-treated neurons, it was not statistically significant. The NO in kainate-treated neurons was reduced most by 69% (P , .01) by PBM.
Conclusion It is often said that PBM has much bigger effects on diseased, sick, damaged, or stressed cells, and comparatively few effects on normal, healthy, undamaged cells. Our data begin to shed some light on this observation. In normal cells at doses around the optimum value of 3 J/cm2 (which incidentally is a fairly broad peak in the dose response curve), PBM will increase the MMP above baseline, accompanied by increases in ATP, intracellular calcium, accompanied by modest increases in ROS and NO. However, if the dose is increased beyond the optimum value (100 times higher in J/cm2) then the MMP is decreased below baseline, the increases in ATP and calcium are lost, and a second peak of ROS and NO is observed. If the cells are subjected to damaging insults such as oxidative stress or excitotoxicity before PBM, then the picture is somewhat different. Because these toxic treatments lower the MMP (sometimes severely) and also lower the ATP levels and reduce cell viability, the effect of PBM is to raise MMP back toward baseline, and to reverse the decreases in ATP levels and cell viability. The effect of ROS depends on what treatment the cells were subjected to. In the case of oxidative stress, the MMP is lowered, and the mitochondria produce substantial amounts of ROS. Note the ROS measured in the oxidative stress cells are not due per se to the oxidizing agents added, but are produced by the damaged mitochondria. When the mitochondria are restored toward normality, the ROS levels come down. The levels of calcium are highly elevated by excitotoxicity due to the opening of ion channels, and PBM can lower these calcium levels possibly by increasing ATP levels back toward normal. The levels of ROS produced as a consequence of excitotoxicity is probably also due to the lowering of MMP by the high calcium levels.
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Neurons are highly metabolic cells, with very active mitochondria compared to many other cell types within the human body. The beneficial activities of PBM may be more pronounced in neurons than they would be with other cells. Perhaps this explains why the brain does appear to respond particularly well to PBM.
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