Novel to the present study, the mechanism of action of PBM utilizing 670 nm light in Müller cells was studied after 24 hours and up to 5 days under high glucose conditions. Our studies showed that as little as 24 hours of high glucose exposure is enough to disrupt mitochondrial function and increase ROS production in Müller glial cells. These early ROS and mitochondrial function changes initiate a cascade of signaling events leading to NFkB activation after 3 days and ICAM-1 and VEGF production. Indeed, treatment with 670nm light attenuated ROS production within an hour and these beneficial effects were also seen in improved mitochondrial function. Daily, short treatment with 670nm light continued to mitigate detrimental signaling effects seen with high glucose alone including reduced NFkB transcriptional activity and decreased production of ICAM-1. Interestingly, we saw no light treatment benefit on the production of VEGF implicating its production by another pathway in addition or in lieu of NFkB. These findings are consistent with other model systems and pathways in which PBM has been shown to be beneficial 9,15.
Oxidative stress has been implicated as an initiating factor in DR pathogenesis 7,22,23,30,31. In cell models of DR, oxidative stress is increased in multiple cell types including human Müller glial cells treated with TBHP 32, RPE grown in high glucose, and RGC grown in high glucose 12. In our study, ROS increases in 24 hours of high glucose treatment. This increase in ROS is mitigated in one hour by one, short light treatment (670nm, 180s). These findings are consistent with others where PBM has been shown to attenuate oxidative stress in many disease models, including DR 8,9, 11–13,33. However, unlike the current study, the majority of these studies have investigated the effects of 670 nm PBM on oxidative stress and ROS production several days after exposure to cytotoxic stressors including high glucose.
Increased oxidative stress can lead to mitochondrial changes. Correspondingly, high glucose causes increased mitochondrial fragmentation in rat Müller glial cells after 7 days in culture. These cells also displayed a change in mitochondrial membrane potential and decreased oxidative phosphorylation and glycolysis 34, which is thought to lead to cell death. In our study, we observed loss of mitochondrial permeability transition pore (MPTP) in as little as 24 hours in high glucose suggesting this is an early event in response to elevated glucose levels. However, we did not investigate the order of insult as oxidative stress can arise from mitochondrial functional loss and mitochondrial functional loss can also lead to increased oxidative stress. Consistent with our study, Zhang et al. showed that a mild oxidative stress (100 mM H2O2) produced an increase in ROS generation, a 30% reduction in mitochondrial metabolic activity and a 20% reduction in mitochondrial membrane potential within 6 hours in primary cultures of human Müller cells and a human cell line (MIO-M1) 32. Thus, acute exposure to either high glucose or to oxidative stress increases ROS generation and disrupts mitochondrial function.
Loss of mitochondrial membrane potential is known to lead to reductions in oxidative phosphorylation 34, we measured cellular ATP content in the presence of high glucose at 24 hours. Although we did observe a trend in a diminution of ATP levels in cells exposed to high glucose medium, this diminution was not statistically significant. It is likely that ATP levels may decrease further at a later time point should the high glucose insult continue. In fact, Devi et al. demonstrates an intracellular ATP decrease of 20% in high glucose conditions (25 mM) compared to normal controls (5.5 mM) 23. Furthermore, PBM may be beneficial as ATP levels trended upward similar to normal glucose levels when cells grown in high glucose were treated with 670nm light.
We observed an increase in NFkB activity at 3 days in culture, likely due to the increased ROS and decreased mitochondrial health. In our study, a 70% increase in NFkB transcriptional activity in the high glucose cultured, sham treated groups was observed in comparison to cells cultured in normal glucose and not receiving treatment (Fig. 2). These findings are consistent with previous work31,35 where increased NFkB activity was observed in response to increased oxidative stress through high glucose in Müller glial cells 31, in monocytes (THP-1) 36 and pericytes 37. Importantly, NFkB concentrations in PBM-treated high-glucose-exposed cultures was not different from that measured in cells maintained under normal glucose. We believe this attenuation is likely due to the decreased ROS and early prevention of mitochondrial changes.
NFkB is a transcription factor that regulates the production of inflammatory and other molecules. In DR, NFkB regulates the production of ICAM-1 and VEGF 12,30,31. Indeed, we observed similar changes in ICAM-1 production as those observed in NFkB activity. In high glucose, Müller glial cells produced 60% more ICAM-1 compared to normal glucose controls, similar to previous studies 31. This increased production was mitigated with PBM. Our in vitro data on the effects of PBM on ICAM-1 concentrations in high glucose-cultured Müller glial cells was comparable to the in vivo effects of PBM in diabetic rodents 11,12. Since ICAM-1 is highly regulated by NFkB, it is likely that for the observed decrease in NFkB transcriptional activity there will be a corresponding decrease in ICAM-1 level 38–40. We measured ICAM-1 levels by Western blot on samples treated for 5 days, based on a prior study by Chen et al. (2012) who observed elevated levels of ICAM-1 when cells were cultured in high glucose for a range of one to seven days 30. After 6 days of in high glucose conditions, a two-fold increase in ICAM-1 concentration relative to levels of GAPDH was observed (Fig. 3). Our findings are in accordance with previous studies. Elevated levels of ICAM-1 were, as well, observed in STZ diabetic mice in comparison to non-diabetic control animals 12,41. Likewise, in a Müller glial cell culture model of DR, a 4-fold increase in ICAM-1 levels was reported for cells cultured in high (25 mM) glucose in comparison to cells cultured in normal glucose conditions for 5 days. Saliba et al. observed an increase in leukostasis, superoxide, and ICAM-1 levels in diabetic mice when compared to non-diabetic control mice. These effects were ameliorated in groups treated with 670 nm LED at a dosage of 6 J/cm2. Importantly, these animals had an improvement in retinal function as measured by ERGs 11. Two months post-induction of diabetes in the STZ diabetic rats the activation of NFkB was increased by 60% in retinal samples compared to non-diabetic counterparts 42. Corresponding to this increase in NFkB activity was a reduction in the antioxidant capacity of superoxide dismutase and an increase in oxidative stress, as measured by the presence of 8-isoprostane in the blood and ratio of GSSG/GSH. 42. The increase in NFkB activity and oxidative stress, as well as the reduction in antioxidant capacity of superoxide dismutase, was partially attenuated in diabetic rats which were treated with the antioxidant resveratrol 42.
Also, VEGF levels increased with high glucose treatment, consistent with many other reports 24,25,43,44. Unexpectedly, we observed no change in VEGF concentration under high glucose conditions compared to high glucose cultured cells treated with 670nm light. This data indicates that in this cell culture model of DR, VEGF and ICAM-1 are not regulated in the same manner. Regulation of VEGF can occur through multiple pathways in addition to NFkB 44. Indeed, Sun et al reports multiple transcriptional proteins which regulate VEGF production including NFkB, p38, TNFa, CREB, vPA, IL-1B, Era, Stat3, and HCAM. Using a primary rat Müller cell model, Sun et al implicate STAT3 in the regulation of VEGF in response to high glucose stimuli. In primary rat Müller cells, VEGF secretion was shown to be controlled by calcium with its transcriptional regulation via CAMKII and HIF1A 43. In neuronal cells, NDMA causes an influx of calcium leading to increased production and release of VEGF 45. Although it is generally accepted that within the injured retina, glial cells are the major source of VEGF 25,45, they are not the only source. In conditional VEGF knockout mice, a Cre/lox system was used to determine the significance of Müller glial cell-derived VEGF. When VEGF synthesis was blocked in Müller glial cells of the conditional mice, a 44.5% decrease in VEGF retinal concentration was observed in comparison to their WT counterparts 25. However, these results also indicate that more than 50% of VEGF is attributed to additional sources, including neuronal cells and RPE cells.
In summary, our findings of the chronic exposure to high glucose show early changes in ROS production and mitochondrial decline, both of which are mitigated with 670nm treatment. Prolonged high glucose culture conditions also lead to NFkB activation and ICAM-1 production. These changes are prevented with 670nm light treatment. Thus, 670nm light can prevent the acute effects of high glucose in a diabetic retinopathy cellular model system and reduce the production of pro-inflammatory mediators, providing a mechanism by which PBM is beneficial.