Glucose Improves the Efficacy of Photobiomodulation in Changing ATP and ROS Levels in Mouse Fibroblast Cell Cultures.

In this study, we tested the idea that photobiomodulation-the application of red to near infrared light (~λ = 600-1300 nm) to body tissues-is more effective in influencing cell metabolism when glucose is readily available. To this end, we used a mouse fibroblast (L-929) cell culture model and had two sets of conditions: non-stressed (10% FBS (foetal bovine serum)) and stressed (1% FBS), both either with or without glucose. We treated (or not) cells with photobiomodulation using an 810 nm laser at 15 mW/cm2 (~7.2 J/cm2). Our results showed that photobiomodulation was neither cytotoxic nor effective in enhancing measures of cell viability and proliferation, together with protein levels in any of the cell cultures. Photobiomodulation was, however, effective in increasing adenosine triphosphate (ATP) and decreasing reactive oxygen species (ROS) levels and this was-most importantly-only in conditions where glucose was present; corresponding cultures that did not contain glucose did not show these changes. In summary, we found that the benefits of photobiomodulation, in particular in changing ATP and ROS levels, were induced only when there was glucose available. Our findings lay a template for further explorations into the mechanisms of photobiomodulation, together with having considerable experimental and clinical implications.

The effect of photobiomodulation on the brain during wakefulness and sleep.

Over the last seventy years or so, many previous studies have shown that photobiomodulation, the use of red to near infrared light on body tissues, can improve central and peripheral neuronal function and survival in both health and in disease. These improvements are thought to arise principally from an impact of photobiomodulation on mitochondrial and non-mitochondrial mechanisms in a range of different cell types, including neurones. This impact has downstream effects on many stimulatory and protective genes. An often-neglected feature of nearly all of these improvements is that they have been induced during the state of wakefulness. Recent studies have shown that when applied during the state of sleep, photobiomodulation can also be of benefit, but in a different way, by improving the flow of cerebrospinal fluid and the clearance of toxic waste-products from the brain. In this review, we consider the potential differential effects of photobiomodulation dependent on the state of arousal. We speculate that the effects of photobiomodulation is on different cells and systems depending on whether it is applied during wakefulness or sleep, that it may follow a circadian rhythm. We speculate further that the arousal-dependent photobiomodulation effects are mediated principally through a biophoton - ultra-weak light emission - network of communication and repair across the brain.

Shifting patterns of cellular energy production (adenosine triphosphate) over the day and key timings for the effect of optical manipulation.

Mitochondria are optically responsive organelles producing energy for cell function via adenosine triphosphate (ATP). But ATP production appears to vary over the day. Here we use Drosophila melanogaster to reveal daily shifts in whole animal ATP production in a tight 24 hours' time series. We show a marked production peak in the morning that declines around midday and remains low through afternoon and night. ATP production can be improved with long wavelengths (>660 nm), but apparently not at all times. Hence, we treated flies with 670 nm light to reveal optimum times. Exposures at 670 nm resulted in a significant ATP increases and a shift in the ATP/adenosine diphosphate (ADP) ratio at 8.00 and 11.00, whilst application at other time points had no effect. Hence, light-induced ATP increases appear limited to periods when natural production is high. In summary, long wavelength influences on mitochondria are conserved across species from fly to human. Determining times for their administration to improve function in ageing and disease are of key importance. This study progresses this problem.

Aged complement factor H knockout mice kept in a clean barriered environment have reduced retinal pathology.

Age-related macular degeneration (AMD) is the largest cause of visual loss in those over 60 years in the West and is a condition increasing in prevalence. Many diseases result from genetic/environmental interactions and 50% of AMD cases have an association with polymorphisms of the complement system including complement factor H. Here we explore interactions between genetic predisposition and environmental conditions in triggering retinal pathology in two groups of aged complement factor H knock out (Cfh(-/-)) mice. Mice were maintained over 9 months in either a conventional open environment or a barriered pathogen free environment. Open environment Cfh(-/-) mice had significant increases in subretinal macrophage numbers, inflammatory and stress responses and reduced photoreceptor numbers over mice kept in a pathogen free environment. Hence, environmental factors can drive retinal disease in these mice when linked to complement deficits impairing immune function. Both groups of mice had similar levels of retinal amyloid beta accumulation. Consequently there is no direct link between this and inflammation in Cfh(-/-) mice.

The eyes of the deep diving hooded seal (Cystophora cristata) enhance sensitivity to ultraviolet light.

The mammalian visual range is approximately 400-700 nm, although recent evidence suggests varying ultraviolet (UV) extensions in diverse terrestrial species. UV sensitivity may have advantages in the dim, blue light shifted environment experienced by submerged marine mammals. It may also be advantageous when seals are on land as UV is reflected by snow and ice but absorbed by fur, enhancing visual contrast. Here we show that the pelagic hooded seal (Cystophora cristata) has a highly UV permissive cornea and lens. Seals like other carnivores have a tapetum lucidum (TL) reflecting light back through the retina increasing sensitivity. The TL in this seal is unusual being white and covering almost the entire retina unlike that in other carnivores. Spectral reflectance from its surface selectively increases the relative UV/blue components >10 times than other wavelengths. Retinal architecture is consistent with a high degree of convergence. Enhanced UV from a large TL surface with a high degree of retinal convergence will increase sensitivity at a cost to acuity. UV electrophysiological retina responses were only obtained to dim, rod mediated stimuli, with no evidence of cone input. As physiological measurements of threshold sensitivity are much higher than those for psychophysical detection, these seals are likely to be more UV sensitive than our results imply. Hence, UV reflections from the TL will afford increased sensitivity in dim oceanic environments.

Topical cyclodextrin reduces amyloid beta and inflammation improving retinal function in ageing mice.

Retinal ageing results in chronic inflammation, extracellular deposition, including that of amyloid beta (Aß) and declining visual function. In humans this can progress into age-related macular degeneration (AMD), which is without cure. Therapeutic approaches have focused on systemic immunotherapies without clinical resolution. Here, we show using aged mice that 2-Hydroxypropyl-ß-cyclodextrin, a sugar molecule given as eye drops over 3 months results in significant reductions in Aß by 65% and inflammation by 75% in the aged mouse retina. It also elevates retinal pigment epithelium specific protein 65 (RPE65), a key molecule in the visual cycle, in aged retina. These changes are accompanied by a significant improvement in retinal function measured physiologically. 2-Hydroxypropyl-ß-cyclodextrin is as effective in reducing Aß and inflammation in the complement factor H knockout (Cfh(-/-)) mouse that shows advanced ageing and has been proposed as an AMD model. ß-cyclodextrin is economic, safe and may provide an efficient route to reducing the impact of retinal ageing.

Systemic administration of Abeta mAb reduces retinal deposition of Abeta and activated complement C3 in age-related macular degeneration mouse model.

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the Western world. There are effective treatments for the vascular complications of neo-vascular AMD, but no effective therapies are available for the dry/atrophic form of the disease. A previously described transgenic CFH-gene deficient mouse model, (cfh-/-), shows hallmarks of early AMD. The ocular phenotype has been further analysed to demonstrate amyloid beta (Aß) rich basement membrane deposits associated with activated complement C3. Cfh-/- mice were treated systemically in both prophylactic and therapeutic regimes with an anti-Aß monoclonal antibody (mAb), 6F6, to determine the effect on the cfh-/- retinal phenotype. Prophylactic treatment with 6F6 demonstrated a dose dependent reduction in the accumulation of both Aß and activated C3 deposition. A similar reduction in the retinal endpoints could be seen after therapeutic treatment. Serum Aß levels after systemic administration of 6F6 show accumulation of Aß in the periphery suggestive of a peripheral sink mechanism. In summary, anti-Aß mAb treatment can partially prevent or reverse ocular phenotypes of the cfh-/- mouse. The data support this therapeutic approach in humans potentially modulating two key elements in the pathogenesis of AMD - Aß and activated, complement C3.

Complement component C3 plays a critical role in protecting the aging retina in a murine model of age-related macular degeneration.

Complement component C3 is the central complement component and a key inflammatory protein activated in age-related macular degeneration (AMD). AMD is associated with genetic variation in complement proteins that results in enhanced activation of C3 through the complement alternative pathway. These include complement factor H (CFH), a negative regulator of C3 activation. Both C3 inhibition and/or CFH augmentation are potential therapeutic strategies in AMD. Herein, we examined retinal integrity in aged (12 months) mice deficient in both factors H and C3 (CFH(-/-).C3(-/-)), CFH alone (CFH(-/-)), or C3 alone (C3(-/-)), and wild-type mice (C57BL/6). Retinal function was assessed by electroretinography, and retinal morphological features were analyzed at light and electron microscope levels. Retinas were also stained for amyloid ß (Aß) deposition, inflammation, and macrophage accumulation. Contrary to expectation, electroretinograms of CFH(-/-).C3(-/-) mice displayed more severely reduced responses than those of other mice. All mutant strains showed significant photoreceptor loss and thickening of Bruch's membrane compared with wild-type C57BL/6, but these changes were greater in CFH(-/-).C3(-/-) mice. CFH(-/-).C3(-/-) mice had significantly more Aß on Bruch's membrane, fewer macrophages, and high levels of retinal inflammation than the other groups. Our data show that both uncontrolled C3 activation (CFH(-/-)) and complete absence of C3 (CFH(-/-).C3(-/-) and C3(-/-)) negatively affect aged retinas. These findings suggest that strategies that inhibit C3 in AMD may be deleterious.

Vitamin D rejuvenates aging eyes by reducing inflammation, clearing amyloid beta and improving visual function.

Vitamin D(3) plays a key role in immune regulation and may protect against the aging process. A focal point for age-related changes is the outer retina of the eye where there is high metabolic demand resulting in a gradual increase in extracellular deposition, inflammation, and cell loss giving rise to visual decline. Here, we demonstrate that vitamin D(3) administration for only 6 weeks in aged mice significantly impacts on this aging process. Treated mice showed significant reductions in retinal inflammation and levels of amyloid beta (Aß) accumulation, which is a hallmark of aging. They also had significant reductions in retinal macrophage numbers and marked shifts in their morphology. These changes were reflected in a significant improvement in visual function, revealing that vitamin D(3) is a route to avoiding the pace of age-related visual decline. Excess amyloid beta deposition and inflammation are risk factors leading to age-related macular degeneration (AMD), the largest cause of blindness in those older than 50 years in developed countries. Recently, vitamin D(3) has been linked epidemiologically to protection against age-related macular degeneration. Hence, vitamin D(3) enrichment is likely to represent a beneficial route for those at risk.

Viewing ageing eyes: diverse sites of amyloid Beta accumulation in the ageing mouse retina and the up-regulation of macrophages.

BACKGROUND: Amyloid beta (Aß) accumulates in the ageing central nervous system and is associated with a number of age-related diseases, including age-related macular degeneration (AMD) in the eye. AMD is characterised by accumulation of extracellular deposits called drusen in which Aß is a key constituent. Aß activates the complement cascade and its deposition is associated with activated macrophages. So far, little is known about the quantitative measurements of Aß accumulation and definitions of its relative sites of ocular deposition in the normal ageing mouse. METHODOLOGY/PRINCIPAL FINDINGS: We have traced Aß accumulation quantitatively in the ageing mouse retina using immunohistochemistry and Western blot analysis. We reveal that it is not only deposited at Bruch's membrane and along blood vessels, but unexpectedly, it also coats photoreceptor outer segments. While Aß is present at all sites of deposition from 3 months of age, it increases markedly from 6 months onward. Progressive accumulation of deposits on outer segments was confirmed with scanning electron microscopy, revealing age-related changes in their morphology. Such progress of accumulation of Aß on photoreceptor outer segments with age was also confirmed in human retinae using immunohistochemistry. We also chart the macrophage response to increases in Aß showing up-regulation in their numbers using both confocal laser imaging of the eye in vivo followed by in vitro immunostaining. With age macrophages become bloated with cellular debris including Aß, however, their increasing numbers fail to stop Aß accumulation. CONCLUSIONS: Increasing Aß deposition in blood vessels and Bruch's membrane will impact upon retinal perfusion and clearance of cellular waste products from the outer retina, a region of very high metabolic activity. This accumulation of Aß may contribute to the 30% reduction of photoreceptors found throughout life and the shortening of those that remain. The coating of Aß on outer segments may also have an impact upon visual function with age.

Held under arrest: Many mature albino RPE cells display polyploidal features consistent with abnormal cell cycle retention.

A population of peripheral retinal pigment epithelium (RPE) cells in mature pigmented rats are retained in the cell cycle and divide, as determined by Ki67 and BrdU labeling. Their cell cycle rate is approximately 5 days. Ten times as many Ki67 positive cells are found in albinos compared with pigmented animals, but it is not known if they actually divide or only express cell cycle markers. In spite of the increased number of cells expressing cell cycle markers, we show here using BrdU, that levels of cell division in albino RPE are similar to those in pigmented animals and have a similar cell cycle rate. Hence, cell cycle activity does not progress through to cell division in the majority of albino RPE cells. Peripheral RPE cells in albinos are different from those in pigmented animals. Many have very large distorted or highly fragmented nuclei. These data along with patterns of Ki67 and BrdU labeling are consistent with such cells being retained abnormally in the cell cycle, replicating their DNA, but not able to progress through to full cell division. Hence, there are two populations of RPE cells in albinos, those undergoing normal cell division and those that appear to be arrested in the cell cycle. These abnormalities are present from early postnatal stages.