SB holds a non-remunerative relation with HN P/L, (hortusnovus.it) which supports research. JS is a director of CSCM Pty Ltd. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: FDM SR JS SB. Performed the experiments: FDM SR MDP SS. Analyzed the data: FDM MDP SR LC LF. Contributed reagents/materials/analysis tools: JS SB. Contributed to the writing of the manuscript: FDM JS SB. Care and welfare of animals: SS.
The central nervous system undergoing degeneration can be stabilized, and in some models can be restored to function, by neuroprotective treatments. Photobiomodulation (PBM) and dietary saffron are distinctive as neuroprotectants in that they upregulate protective mechanisms, without causing measurable tissue damage. This study reports a first attempt to combine the actions of PBM and saffron. Our working hypothesis was that the actions of PBM and saffron in protecting retinal photoreceptors, in a rat light damage model, would be additive. Results confirmed the neuroprotective potential of each used separately, but gave no evidence that their effects are additive. Detailed analysis suggests that there is actually a negative interaction between PBM and saffron when given simultaneously, with a consequent reduction of the neuroprotection. Specific testing will be required to understand the mechanisms involved and to establish whether there is clinical potential in combining neuroprotectants, to improve the quality of life of people affected by retinal pathology, such as age-related macular degeneration, the major cause of blindness and visual impairment in older adults.
The central nervous system in mammals has only a limited ability to repair its neuronal circuitry. Its functional stability is achieved by ensuring the stability of individual neurons and by redundancy that enables normal function despite substantial loss of neurons. Age-related loss of retinal stability results in diseases such as age related macular degeneration (AMD).
Inflammation is an important feature of the aged retina and in many retinal diseases, including AMD. Recent studies have demonstrated that exposure to 670 nm light reduces inflammation in the retina undergoing degeneration
Saffron has been used for a long time in traditional medicine. Its effectiveness as a neuroprotectant was pioneered by Maccarone and colleagues
Microarray analysis
Our previous study has described the time course of protection for dietary saffron and photobiomodulation (PBM), in an animal model of light damage
All experiments conducted were in accordance with the policies of the Association for Research in Vision and Ophthalmology (ARVO) and with the approval of the Animal Ethics Committee at the University of Sydney (Approval number: K22/5-2009/2/5003). Animals were raised and experiments conducted in cyclic 5 lux light (12 hrs: 12 hrs). Adult Sprague Dawley (SD) albino rats were born and raised in dim cyclic light conditions (12 h at 5 lux, 12 h dark)
Light damage (LD) was generated by exposing the animals to 1000 lux light for 24 h. The light was generated by fluorescent tubes located above the cage. For the exposure period, the animals were provided with food and water from containers on the floor of the cage, to ensure consistent exposure to the light. After LD the animals were returned to dim cyclic illumination for a post-exposure period of one week (1 w). The animals were euthanized (Lethobarb 60 mg/kg intraperitoneal) and retinal tissues were obtained for analysis.
Five groups of animals were used:
Control: These animals (n = 4) were raised in 5 lux cyclic light, as above.
Light damaged (LD) control: These animals (n = 10) were raised in dim cyclic light, then exposed to bright light for 24 h, and returned to dim cyclic light for 1 w.
Saffron-conditioned LD: These animals (n = 10) were raised in dim cyclic light and, prior to exposure to bright light, were preconditioned for 10 days with saffron at 1 mg/kg/day. Saffron (stigmata of
Photobiomodulation (PBM) conditioned LD: These animals (n = 10) were raised in dim cyclic light, exposed to bright light and kept for a further week, as above. For 7d prior to exposure to the bright light, each animal was exposed to 670 nm red light from a WARP 75 source (Quantum Devices Inc, Barneveld, WI, USA). Animals were gently restrained under a plexiglass platform with the eyes ∼2.5 cm below the platform. The WARP 75 device was placed on top of the platform and turned on for 3 min. This arrangement provided a fluence of 4.0–4.5 J/cm2 at the eye, calculated from an estimate of power at 2.5 cm from the LED array, made using a calibrated sensor provided by Quantum Devices (Barneveld, Wisconsin). The animals did not hide from or appear agitated by the red light.
Combined conditioned: These animals (n = 10) were raised in dim cyclic light, exposed to bright light and kept for a further week, as above. For 10d prior to the exposure to bright light, each was exposed simultaneously to both the saffron and the PBM conditioning described above.
The superior aspect of the eye was marked with an indelible marker by a stitch in the conjunctiva, after anaesthesia and prior to euthanasia. After euthenasia, the eyes were dissected free and fixed by immersion in 4% paraformaldehyde fixative buffer at 4°C for 1 h. After three rinses in 0.1 M phosphate-buffered saline (PBS), eyes were left overnight in a 15% sucrose solution to provide cryoprotection. Eyes were embedded in mounting medium (Tissue Tek OCT compound; Sakura Finetek, Torrance, CA) by snap freezing in liquid nitrogen. Cryosections were cut at 20 µm (CM1850 Cryostat; Leica, Wetzlar, Germany) with the eyes oriented so that the sections extended from superior to inferior edge. Sections were mounted on gelatin and poly-L-lysine-coated slides and were then dried overnight in 50°C oven and stored at −20°C until processed.
Sections were labelled for apoptotic cell death using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technique
Retinal sections were washed with 0.1 M PBS (10 min twice) and incubated in 10% normal goat serum in 0.1 M PBS for 1 hour at room temperature, to block non-specific binding. Sections were then incubated overnight at 4°C in rabbit polyclonal anti GFAP (1∶700; DakoCytomation, Campbellfield, Australia). After 3 rinses in PBS for 10 minutes each, sections were incubated with an appropriate secondary antibody (1∶1.000 ALEXA Fluor 594; Molecular Probes, Invitrogen Carlsbad, CA), for 1 h at room tempertaure
Three measures of neuroprotection were used, the surviving population of photoreceptors, the rate of photoreceptor death, and the expression of the stress-inducible protein GFAP in Müller cells. All were assessed 1 w after exposure to damaging light.
We estimated photoreceptor survival by measuring the thickness of the outer nuclear layer (ONL). Specifically, we recorded the ratio of the thickness of the ONL to the thickness of the retina (from the inner to the outer limiting membrane), measured at 0.40 mm intervals, from the superior to the inferior edge of the retina. The ratio of ONL to retinal thickness was used as a measure of ONL thickness, rather than the absolute thickness of the ONL (µm), to compensate for oblique sectioning.
We measured the length of Müller cells along which GFAP expression was evident (µm), as a proportion of retinal thickness (from the inner to the outer limiting membrane), This was recorded at 0.4 mm steps along retinal sections, from the superior to the inferior edge. Measurements were made in at least 2 sections from one eye of each animal studied.
Electroretinograms (ERGs) were recorded in control and treated animals 1 day before and 1 week after high intensity light exposure (light damage LD). Albino rats were previously dark adaptated overnight. Ketamine : xylazine anaesthesia was used with intra peritoneal injection of 100 mg/kg ketamine, 12 mg/kg xylazine (Ketavet 100 mg/ml, Intervet production srl; Xylazine 1 g, Sigma Co.). Corneas were anesthetized with a drop of novocaine, and pupils were dilated with 1% atropine sulfate (Allergan, Westport, IR). Body temperature was maintained at 37±0.5°C with a heating pad controlled by a rectal temperature probe. Recordings were made from the left eyes, with a gold electrode loop (2 mm in diameter) placed on the cornea while the right eye was fully covered with a bandage. The reference electrode was placed on the right cornea under the bandage, and the ground electrode was inserted in the anterior scalp, between the eyes. The rat's head was positioned just inside the opening of the Ganzfeld dome (Biomedica Mangoni, Pisa, Italy). This electronic flash unit generated flashes of a range of intensities from 0.001–100 cd/m2. Responses were recorded over 300 ms plus 25 ms of pre-trial baseline, amplified differentially, bandpass filtered at 0.3 to 300 Hz, digitized at 0.25- to 0.3-ms intervals by a personal computer interface (LabVIEW 8.2; National Instruments, Milan, Italy), and stored on a disc for processing. Responses from several trials were averaged (
The significance of differences in ONL thickness and GFAP labelling associated with conditioning were assessed using ANOVA, followed by a Tukey test. The Tukey test was used for all pairwise comparisons of the mean values. Results are expressed as the mean ± SE.
In control retina (dim-reared, not exposed to bright light, unconditioned by saffron or PBM) the rate of photoreceptor death, assessed by the frequency of TUNEL+ cells in the ONL (
We tested whether the number of TUNEL+ cells were significantly different among 5 experimental groups. In all three treated groups, the number of TUNEL+ profiles was significantly smaller than in the light damage (LD) group. The histogram bars show mean numbers of TUNEL+ cells/mm ONL, for each experimental group; the error bars show standard error of the means. Statistical significance indicator: (***) p<0.001.
A–E: Representative bisbenzimide labelling in control (A), light damage (B), 7 days PBM (C), 10 days saffron (D) and combined treatment (E) groups, 1 w after light damage. Images are taken one millimeter dorsal from optic disc. F: The ratio of ONL thickness to retinal thickness from the superior to the inferior edge. The arrow shows the position of the optic disc. Different symbols represent different experimental groups. For each group, each point shows the mean of the group; the error bars show standard errors of the mean. G: The ratio of ONL thickness to retinal thickness, in the hot spot area (the area of greatest light-induced damage), 1 mm superior to the optic disc. For each group, each point shows the mean of the group; the error bars show standard errors of the mean. Statistical significance indicators: (***) p<0.001; (**) p<0.01, for the difference of each group from the light damage group value.
The result is shown quantitatively in
A–E: Representative GFAP labelling in control (A), light damage (B), 7 d PBM (C), 10 d saffron (D) and combined treatment (E) groups, 1 w after light damage. Light damage induced the up-regulation of GFAP in the radially oriented Müller cells; the protein is visible along the full length of the Müller cells, from the ILM to the OLM. F: Length of the Müller processes expressing GFAP as a function of distance from superior edge in the five experimental groups. The arrow shows the position of the optic disc. Each point shows the mean labelled length; error bars show standard deviations of the mean. G: Mean length of Müller processes expressing GFAP, averaged across superior retina, in: Control (A), Light Damage (B), 7 days PBM (C), 10 days saffron (D) and combined (E) groups. In all treated groups, the length of Müller cells expressing GFAP was less than in the light damage group. The error bars show standard errors of the mean. Statistical significance indicator: (***) p<0.001.
The result is shown quantitatively in
A: ERG b-wave amplitude (µV) as a function of flash brightness (cd/m2) in four experimental groups. As for other data, each point represents the group mean and error bars show standard deviations of the mean. B: The amplitude of the b-wave normalized to control values in the 5 experimental groups. Histogram bars show means for each experimental group; error bars show standard deviations of the mean. The comparisons were made from data obtained at a fixed value of luminance (10 cd/m2) before saturation. Single treatment with saffron and PBM mitigated the b-wave reduction induced by light damage, and the differences between treated and untreated groups were significant. Combined treatment also mitigated the reduction of the b-wave, but the difference was not statistically significant. C: Representative traces for each group to a stimulus flash of 10 cd/m2 of luminance. The error bars show standard errors of the mean. Statistical significance indicators: (***) p<0.001; (**) p<0.01, (*) p<0.05.
The difference in the amplitude of the b-wave is significant between unconditioned and PBM conditioned light damaged group (
The present observation, that neuroprotective effects of saffron and PBM are not additive, does not support our working hypothesis, formulated on the basis of results obtained in a microarray study
The experimental stress we used (bright continuous light exposure) induces oxidative stress, to which neurons are vulnerable. Oxidative stress can also contribute to tissue damage indirectly, by activating pathways that induce the expression of stress sensitive genes, and by glia-mediated inflammation that causes secondary neuronal damage
In both the ONL and GFAP data, combined treatment gave a poorer outcome than in single saffron conditioning; the differences were statistically significant. This suggests a limited degree of interference between the two forms of conditioning. Confirmation of this interference and more knowledge of the underlying mechanisms seem necessary for progress towards the successful combination of neuroprotectants. The suggestion of interference gives weight to the main outcome of this study, that the protective effects of PBM and saffron are not additive; greater protection is not gained when both forms of conditioning are applied.
More positively, our results show that combination of saffron and PBM preconditioning does not introduce major side effects or induce a major reduction in neuroprotection. The general idea of combining different treatments to reach a better results remains valid, but needs further investigation to form a basis in animal models for human trials of combinations of protectants.
We wish to thank Ms. Charean Adams (Bosch institute, University of Sydney) for her assistance and support throughout the execution of the experiments and Dr. Rita Maccarone (University of L'Aquila) for her support with electroretinographic recordings.