light intensities presented in ascending order, beginning below threshold, to record the b-wave sensitivity curves and allow calculation of the saturated b-wave amplitude (Bmax). B-wave amplitude was measured between a- and b-wave peaks for quantitative analysis.

42.2.5Morphological Evaluation of Photoreceptor Rescue by Quantitative Histology

Animals were killed by an overdose of carbon dioxide after electroretinographic testing. The eyes were enucleated, fixed, embedded in paraffin, and 5 μm think sections were cut along the vertical meridian. In each of the superior and inferior hemispheres, outer nuclear layer (ONL) thickness was measured at nine defined points as described (Li et al. 2006; Kong et al. 2006). In each of the experiments where ONL thickness was quantified, a single section from each of 8 eyes was measured.

42.2.6 Statistical Analysis

Results are expressed as mean±SD. Differences were assessed by one-way ANOVA and by t test. A P value less than 0.05 was considered significant.

42.3 Results

42.3.1 LED Attenuated the Light Damage Area in Retinas

The retinas, especially the outer nuclear layer, did not alter in the control, LED control, and light damage groups of 900 lux with or without LED protection (Fig. 42.1a–d). The extent of the degeneration retinas, explored to constant light (1,800 lux) for 3 h, decreased from approximately 1/2 to 1/6 after LED protections (Fig. 42.1e, f). Performing, a continue section cutting along the vertical meridian, we found that the damage extension were within a small circle, the center of which seems at the vertical meridian through the optic nerve head shown in Fig. 42.2. In most cases in group F, the damage region is located in the superior retina.

42.3.2 LED Protected the Morphology of Light Damage Retina

The extent of photoreceptor degeneration in rats was evaluated by measuring the thickness of outer nuclear layers (ONL) (Fig. 42.3). The area under every line stood for the amount of survival cells in ONL of each retina. Thus, the area between every line and the control one stood for the amount of lost cells in ONL induced by light

Fig. 42.1 The extent of light-induced retina damage. (a) Untreated control (b) LED control (c,e,g) 3 h constant light damage of different illuminations of 900, 1,800 and 2,700 lux, respectively. (d,f,h) 3 h constant light damage of different illuminations of 900, 1,800 and 2,700 lux, respectively+LED treatment for 30 min, 3 h before the light damage and 0, 24 and 48 h after the light damage. (a d) Without significant morphologic alteration in retinae. (e h) the black double arrow indicates the light-induced damage area, in which the outer nuclear layer (ONL) becomes thinner

Fig. 42.2 Two-dimensional schematic diagram of light-induced retina damage area. (a), Untreated control. (e), 3 h constant light damage of illuminations of 1,800 lux, (f) 3 h constant light damage of illuminations of 1,800 lux, +LED treatment for 30 min, 3 h before the light damage and 0, 24 and 48 h after the light damage

damage. There was a significant difference of lost cell between group E and LEDprotected F (p < 0.01), both of which were exposed to illumination of 1,800 lux for 3 h. However, there was no significant difference between group G and LEDprotected H (p > 0.05), which were both of 2,700 lux. Then, an interesting event

Fig. 42.3 A measurements of ONL thickness of the light-induced damage retina

emerged: the amount of lost cells in superior and inferior retina was significantly different in group E (p < 0.01) and F (p < 0.01), however, not in other groups.

The high magnification pictures were provided for group E and F. Three hours of exposure to constant light (1,800 lux) reduced the thickness of the ONL of photorecepor cell nuclei from the normal 10–13 rows in control animal (Fig. 42.4a) to 2–3 rows (Fig. 42.4e, f) in the most severe degenerated region of the retinas, while the IS and OS layer disappear simultaneously. However, with the LED protection (Fig. 42.4f), there was significant rescue of photoreceptors with the ONL having 2–5 rows of nuclei.

42.3.3 LED Protected the Function of Light Damage Retina

The effect of LED-treatment on retinal function was determined by electroretinography 5 days after exposure to constant light for 3 h. Histograms of Bmax values are presented in Fig. 42.5. The Bmax did not alter in the LED control and light damage groups of 900 lux (with or without LED protection) compared with the untreated control. Exposure to illumination of 1,800 lux obviously reduced the b-wave amplitudes to 40% in group E. There was a significant difference between group E and F,

42.4 Discussions

Results of this study demonstrate the therapeutic benefit of LED irradiation in the survival and functional recovery of the retina in vivo after light-induced damage. We provide in vivo evidence that 670 nm LED photo-irradiation reduces the retinal lesion by light microscope and quantitative histology, and attenuates the damage of retinal function in rod and cone pathways by measuring the ERG b wave. The mechanism of LED irradiation emphasizes to the organism absorption of near-infrared light and subsequent cellular biochemical alteration, obviously, it is different from the ‘light precondition’ reported by Liu et al. (1998). Liu et al. (1998) found that the preconditioning with bright light, that could help the animal resistant to subsequent light damage, by evoking two opposing processes: a fast degenerative process and a relatively slower protective process, which needs 2 days to develop fully. We gave the LED treatment 3 h before the light damage, however it is too short to develop the protective process. Besides, the power intensity of led in present study is 50 mW/cm2, which is not as bright as described in Liu C group’s article, 115–130 cd.

Despite the cellular mechanisms underlying LED treatment for light damage remain uncertain, the junction of both current theory about the LED and light damage may give us a reasonable explanation. The continued research efforts have provided a wealth of information on LED treatment and light damage. Gene discovery studies conducted using microarray technology documented a significant up-regulation of gene expression in pathways involved in mitochondrial energy production and antioxidant cellular protection (Wong-Riley et al. 2005). Britton Chance’s group reported about 50% of near-infrared light (NIL) is absorbed by mitochondrial chromophores such as cytochrome c oxidase (Beauvoit et al. 1994).Cytochrome oxidase is an integral membrane protein and contains four redox active metal centers: the dinuxlear CuA, CuB, heme a and heme a3, all of which have absorbance in the red to near-infrared range detectable in vivo near-infrared spectroscope (Cooper and Springett 1997). Cytochrome c oxidase is the terminal enzyme of the electron transport system of all eukaryotes (Krab and Wikström 1987), oxidizing its substrate cytochrome c and reducing molecular oxygen including free radical oxygen to water. Meanwhile, it is an important energy-generating enzyme critical for the proper functioning of almost all cells, especially those of highly oxidative organs and tissue such as the brain and retina.

Recent observations demonstrate the loss of calcium homeostasis, free radical damage, and any other processes lead to mitochondrial failure in light damage process (Barron et al. 2001; Missiaen et al. 2000; Carmody et al. 1999). Penn et al. (1987) have reported an increased antioxidant levels in the animal retinas, which were raised in a bright rearing environment. He implied the antioxidants could ameliorate light-induced retinal degeneration, suggesting a role for oxidative stress in photoreceptor cell death. Carmody RJ group’s study (Carmody et al. 1999) demonstrates an early and sustained increase in intracellular reactive oxygen species accompanied by a rapid depletion of intracellular glutathione in an in vitro model of photoreceptor apoptosis. Those early changes in the cellular redox state lead to a disruption of mitochondrial transmembrane potential, ultimately result in