1.2. Mechanisms of PBM
It is the first law of photobiology that a photon must be absorbed by a specific molecular chromophore in order to have any biological effect. The chromophores that have been postulated to be useful in PBM, absorb at different wavelength regions of the electromagnetic spectrum (blue, green, red, NIR), and are shown in Figure 1
and discussed below.
Cytochrome C oxidase (CCO) is the terminal enzyme (unit IV) in the electron transport chain situated in the outer mitochondrial membrane. The electron transport chain, through a series of redox reactions, facilitates the transfer of electrons across the inner membrane of the mitochondria. The net result of these electron transfer steps is to produce a proton gradient across the mitochondrial membrane that drives the activity of ATP synthase (sometimes called unit V) that produces the high-energy adenosine triphosphate (ATP) from ADP. CCO mediates the transfer of electrons from cytochrome C to molecular oxygen. CCO is a complex protein, composed of thirteen different polypeptide sub-units, and also contains two heme centers and two copper centers. Each of these heme and copper centers can be either oxidized or reduced, giving sixteen different oxidation states. Each of these oxidation states has a slightly different absorption spectrum, but CCO is almost unique amongst biological molecules in having a significant absorption in the near-infrared spectrum. In fact, Britton Chance estimated that over 50% of the absorption of NIR light by biological tissue could be attributed to this single enzyme as a chromophore [5
In many publications, CCO has been shown to be a biological photoacceptor and transducer of signals activated by light in the red and NIR regions of the spectrum [6
]. Specifically, absorption of the photons delivered in PBM, seems to promote an increase in the availability of electrons for the reduction of molecular oxygen in the catalytic center of CCO, increasing mitochondrial membrane potential (MMP), and increasing levels of ATP, cyclic adenosine monophosphate (cAMP), and reactive oxygen species (ROS), all of which indicate increased mitochondrial function, and can trigger initiation of cellular signaling pathways [8
]. However recently, the CCO hypothesis has been brought into question. Lima et al. [9
] genetically engineered two different kinds of cells to not express any active CCO, and found they responded equally well to 660 nm light, compared to wild type cells. Although other units in the electron transfer chain, such as complexes I-IV and succinate dehydrogenase also show increased activity as a result of PBM, CCO is still believed to be one of the primary photoacceptors. This notion is supported by the fact that low-level light irradiation such as PBM causes increased oxygen consumption, and is bolstered by the fact that the majority of oxygen consumption occurs at complex IV, and moreover that the addition of sodium azide, a CCO inhibitor, abrogates the effects of PBM [10
]. Moreover, rho-zero cells that lack functional mitochondria do not respond to PBM, in the same way as their wild-type counterparts [12
Nevertheless, despite the amount of évidence in favor of CCO being a major chromophore for red and NIR light, mounting evidence is suggesting that this is not the whole story. Lima et al. [9
] investigated two cell lines lacking CCO, one mouse line with the Cox10 knocked out (that could not synthesise the heme a cofactor) and a second human line with a mutation in the mtDNA gene coding for tRNA lysine (that lacked three critical CCO subunits). PBM (660 nm) caused increased cell proliferation in both wild type and CCO knock out cells, together with increased ATP and citrate synthase levels. These results showed that functional CCO was not required for its ability to enhance metabolism and cell proliferation.
A recent editorial [13
] from Sommer in Ulm, Germany suggested that the effects of red and NIR light (especially pulsed at low frequency such as 1 Hz) on the interfacial water layers (IWL) inside cells could be an alternative explanation. If these IWL were inside the mitochondria, then the lowering of viscosity as a result of the energy absorption, could allow the molecular rotor, which is ATP synthase, to rotate faster and produce more ATP. On the other hand if the IWL were localized within the plasma membrane, light absorption could increase the uptake of nutrients accounting for increased proliferation.
Regardless of the actual chromophore, PBM can trigger retrograde mitochondrial signaling [14
]. This refers to signals and communications passing from the mitochondria to the nucleus of a cell, rather than vice versa. The aforementioned mitochondrial changes result in an altered mitochondrial ultrastructure, and triggering of mitochondrial biogenesis [15
]. As a result, membrane permeability and ion flux at the cell membrane are altered, in turn leading to the altered activity of activator protein-1 (AP1) and NFκB [16
There is emerging evidence that other primary chromophores such as opsins, flavins and cryptochomes, may mediate the biological absorption of light, particularly at shorter wavelengths (blue and green). Opsins contain a cis
-retinaldehyde molecule as a chromophore that is photo-isomerized to the all-trans isomer, thus producing a change in protein conformation and initiating a signaling cascade [17
]. Flavins and flavoproteins contain a chromophore such as riboflavin, flavin mononucleotiode, or flavin adenine dinucleotide and can carry out redox reactions when excited by light [18
]. Cryptochromes are a special sub-class of flavoproteins that act as blue–light receptors in plants, animals and even humans [19
Although evidence proving that light-gated ion channels can be cited as mechanisms of action in PBM is sparse at the present time, it is gradually increasing. PBM is most likely to affect transient receptor potential (TRP) channels. First discovered in a Drosophila mutant as the mechanism responsible for the vision of insects, they are now known to be sensitive to light [20
], in addition to a wide variety of other stimuli. TRP channels are calcium channels, and are modulated by phosphoinositides [21
]. Light-gated ion channels have attracted immense attention in the field of optogenetics [22
]. However the majority of these studies employ ion channels similar to bacterially derived channelrhodopsin [22
]. The majority of research relating PBM to light-gated ion channels has been done by testing the TRPV “Vanilloid” subfamily of TRP species. Evidence from studies done by various groups [23
] have led to the general consensus that TRP channels are most likely to be activated by green light. However, because green light lacks the same penetrating ability of infrared or near-infrared light, it lacks practical clinical application. However, Ryu et al. found that exposure to infrared (2780 nm) wavelength light attenuated TRPV1 activation, causing a decrease in generation of pain stimuli [24
]. A similar, but far less dramatic antinociceptive effect was also observed when TRPV4 was exposed to light of the same wavelength. TRPV4 was also shown to be responsive to 1875 nm pulsed light, although it cannot be ruled out that the results were due to thermal stimuli rather than light stimuli [25
], as water is the primary absorber of infrared in this region.
It is clear that water must be by far the most important chromophore at infrared wavelengths (>900 nm), considering its molecular absorption coefficient and its relative abundance in cells and tissues. Nevertheless PBM as usually carried out, does not produce excessive heating of the tissues, especially within the brain. In fact the most noticeable heating effect (if any) is felt on the skin of the scalp. How then can we explain that PBM can have powerful effects on the brain at wavelengths as long as 1064 nm [27
]? One answer may lie in the concept of ‘nanostructured water’ or ‘interfacial water’ elaborated by Pollack [29
]. This exclusion zone (EZ) water (which may be the same as the IWL discussed above [13
] absorbs optical radiation which produces distinct physical changes in parameters such as viscosity and pH. Since the EZ water layers occur on intracellular membranes, it is reasonable to suggest that ion channels embedded within these membranes (for instance in mitochondria), may be triggered by these physical changes. Since bulk water does not absorb IR light to the same degree as EZ water, this would explain why biochemical changes can take place within the cells, while there is no detectable bulk heating of the tissue, as would have been expected if the IR energy was absorbed by all water molecules.