Low-intensity far-red light inhibits early lesions that contribute to diabetic retinopathy: in vivo and in vitro

Abstract

Purpose: Treatment with light in the far-red to near-infrared region of the spectrum (photobiomodulation [PBM]) has beneficial effects in tissue injury. We investigated the therapeutic efficacy of 670-nm PBM in rodent and cultured cell models of diabetic retinopathy.

Methods: Studies were conducted in streptozotocin-induced diabetic rats and in cultured retinal cells. Diabetes-induced retinal abnormalities were assessed functionally, biochemically, and histologically in vivo and in vitro.

Results: We observed beneficial effects of PBM on the neural and vascular elements of retina. Daily 670-nm PBM treatment (6 J/cm(2)) resulted in significant inhibition in the diabetes-induced death of retinal ganglion cells, as well as a 50% improvement of the ERG amplitude (photopic b wave responses) (both P < 0.01). To explore the mechanism for these beneficial effects, we examined physiologic and molecular changes related to cell survival, oxidative stress, and inflammation. PBM did not alter cytochrome oxidase activity in the retina or in cultured retinal cells. PBM inhibited diabetes-induced superoxide production and preserved MnSOD expression in vivo. Diabetes significantly increased both leukostasis and expression of ICAM-1, and PBM essentially prevented both of these abnormalities. In cultured retinal cells, 30-mM glucose exposure increased superoxide production, inflammatory biomarker expression, and cell death. PBM inhibited all of these abnormalities.

Conclusions: PBM ameliorated lesions of diabetic retinopathy in vivo and reduced oxidative stress and cell death in vitro. PBM has been documented to have minimal risk. PBM is noninvasive, inexpensive, and easy to administer. We conclude that PBM is a simple adjunct therapy to attenuate the development of diabetic retinopathy.

Keywords: diabetic retinopathy; photobiomodulation; retinal ganglion cells.

Figures

Figure 1.

Far-red PBM therapy significantly reduced diabetes-induced death of cells in the RGC. Rats that had been made experimentally diabetic (D) or remained nondiabetic (N) were exposed to 670-nm light (PBM) each day (4 minutes, 25 mW/cm2, 6 J/cm2), or no treatment. After 10 weeks diabetes, retinal sections were assayed with TUNEL kit to assess death of cells in the RGC layer (n = 5 per experimental group).

Figure 2

(A) PBM for 4 minutes per day (25 mW/cm2, 6 J/cm2) inhibited diabetes-induced decrease in ERG. ERG responses were measured following approximately 2 months of PBM. Recordings on test animals were obtained after dark adaptation overnight using standard methods. Treatment of diabetic animals with PBM partially preserved photopic b wave responses (D is significantly different from N, N+PBM, and D+PBM, all P < 0.05) (n = 5 per experimental group). (B) Representative ERG waveforms (max intensity) under scotopic and photopic conditions are shown.

Figure 3.

Effect of diabetes or 670-nm PBM on CO activity (A, B) or expression (C) in retinas from rats diabetic 10 weeks. (A) Micrograph shows a representative cryosection demonstrating sites of CO activity. Black color indicates more activity. (B) The figure summarizes these reaction intensity results for layers of the retina. N animals are indicated in white bars, and D animals are indicated by gray bars. Cross-hatching to right and left represent PBM daily or 3 days per week, respectively. Asterisk demonstrates P < 0.05 different from D control. In none of the retinal layers was CO activity in diabetic controls significantly different from that in nondiabetic controls. (C) The figure summarizes expression of mitochondrial CO (complex IV subunit I). Expression of the protein did increase in diabetes, but PBM had no significant effect on that change. Representative results from Western blots are shown. (D) The figure indicates that high glucose with or without PBM did not alter CO activity in RGC5 cells in vitro. The graphical data in the figures summarize data from five animals per group (A–C), or six dishes per experimental condition (D).

Figure 4.

Effect of diabetes and far-red PBM on oxidative stress in retina and retinal cells. (A) Diabetes (2 months) increased superoxide production by retina, and this increase was inhibited by 4 minutes per day PBM. Administration of PBM either daily or only three times per week both significantly inhibited the superoxide generation, but daily therapy was more effective. (B) Diabetes caused a significant reduction in expression of MnSOD, which was corrected by the PBM therapy. (C) In contrast to intact retina, neither elevated glucose nor PBM (two sessions of 200 seconds each day) had any effect on MnSOD expression in RGC5. Representative results from Western blots are shown under the graphs. The graphical data in the figures summarizes data from three to six animals per group and six dishes per experimental condition.

Figure 5.

PBM of 670 nm for 4 minutes per day (25 mW/cm2, 6 J/cm2) inhibited the diabetes-induced increase in leukostasis in retinal blood vessels. (A) The number of leukocytes adherent to the capillary walls (after perfusion to remove unbound erythrocytes and white blood cells) is indicated per retina in the figure. (B) Expression of ICAM-1 in retina, the adhesion molecule that leukocytes adhere to, is summarized. Representative results from Western blots are shown. The graphical data in the figures summarizes data from five animals in each experimental group.

Figure 6.

Effects of elevated glucose and 670-nm PBM on RGC5 cells in vitro. Incubation in elevated glucose significantly increased expression of phosphorylated forms of AKT, p38 MAPK, and HSP 27, and downregulated expression of HO-1 (A–D). In each case, PBM (200 seconds, 25 mW/cm2, 5 J/cm2) significantly inhibited these abnormalities toward values seen in normal (5 mM) levels of glucose. The graphical data in the figures summarizes data from six dishes for each experimental condition.