OPTIC FILTERS AGAINST THE PHOTOTOXIC EFFECT OF THE VISIBLE SPECTRUM IN THE RETINA: animal testing

Transcription

1 Trial: Photoxicity and photoprotection: Animal testing OPTIC FILTERS AGAINST THE PHOTOTOXIC EFFECT OF THE VISIBLE SPECTRUM IN THE RETINA: animal testing 1 SUMMARY Title: OPTIC FILTERS AGAINST THE PHOTOTOXIC EFFECT OF THE VISIBLE SPECTRUM IN THE RETINA: animal testing Aims: Evaluating the phototoxic effect of the light, and the protective action of the selective-absorption optic filters against the short wavelengths in lab-animal retinae. Sample: 116 Pigment rabbits Methodology: Sample was divided in two groups according the intraocular lens implanted: Control groups is formed by rabbits without cataract surgery, study group comprised rabbits implanted with a blue light absorbing intraocular lens in the right eye and implanted with a clear intraocular lens in the fellow eye. Rabbits were exposed to white light, white light in absence of blue light (named yellow light) or blue light. Following the exposure, it was realized a structural, genetic and inmunohistological analysis. Results: 1.- The chronic exposure to circadian lighting, with different types of light, causes a fall down in the cell density of the inner and outer nuclear layers, as well as in the ganglionar layer. The greatest loss of retinal cells takes place in the animals submitted to blue light. Exposure to short-wavelength light favors apoptosis, as there is an increase in the expression of the pro-apoptotic gene Bad, a reduction in the expression of the anti-apoptotic gene Bcl-XL, an increase of the expression of some MMPs involved in the onset of drusen and an increase in the expression of TIMP-1 and TIMP-2. The harmful blue-light effects can be prevented absorbing these short wavelengths with selective optic filters. The filtering of the short wavelengths inhibits the start signaling for apoptosis, as it modifies the expression of the protooncogenes c-fos and d-jun. causes an up-expression of the gene Trk-B. This can be a response to the neuroprotective stimuli caused by this molecule. Conclusions: The absorption of the short wavelengths using a selective optic filter is beneficial, and causes the same results irrespective their application on the very light source, or in the implanted intraocular lens. In other words, the short-wavelength filtering effects take place irrespective the filter placing with regards to the retina.

2 1 INTRODUCTION Vision is the sense that allows us detect part of the radiating energy and interpret it. But light itself can cause a toxic effect in the retina of alive individuals, especially the most energetic radiations of the visible spectrum: the violet and blue light. On the other hand, there are selective-absorption filters which manipulate the light lighting different bands of the electromagnetic spectrum. This doctoral thesis tries to prove the efficiency of the selective-absorption filters like a means to neutralize the harmful effect of light on the retina, avoiding the retinal damage-causing changes. In spite of its acknoledged importance, the macular degeneration proccess in alive eyes has not received much attention, mainly because of technical difficulties. It seems especially difficult to quantify the initial distrophy degree, and the development of the tisular damage in the retinal neurons. Only in the last decade the technological innovation in the retinal degeneration diagnosis has enabled the performance of longitudinal and cross, quantitative studies in human and research-animal retinae, especially emphasizing the quantification, to get objective data, and perform thorough comparative analyses. This study has been designed in animal testing with the aim to shorten the time to get results, in order to conceptually extrapolate them in humans, prior studies to clinical trials. The reason for this work is to collaborate in the learning of the causes, necessary treatments, and palliative actions regarding the retinopathies. 2 AIMS 2.1 GENERAL AIM Evaluating the phototoxic effect of the light, and the protective action of the selectiveabsorption optic filters against the short wavelengths in lab-animal retinae. 2.2 SPECIFIC AIMS Structural analysis of the retina 1.- Performing a quantitative analysis of the different nuclear strata in the retina, as start-point for being indicative of the retinal layers more affected. 2.- Comparing the cell density of the retinal inner and outer nuclear layers of rabbits exposed to light with different spectral composition. The animals will be submitted to a mixed implant of intraocular lenses with different absorbance in each eye (yellow/clear). 3.- Determining whether the light induces cell death by apoptosis, or there are other mechanisms involved in the cell light-induced loss. 4.- Analizing the protective effect of the selective-absorption optic filters against the most energetic light-spectrum bands in the changes along the retinal cell death process. 5.- Determining what is the visual photoreceptor type most affected by the different kinds of lighting. 2

3 6.- Evaluating the effect of intraocular lenses with a selective blue-light filter regarding the type of affected photoreceptor Analysis of the gene expression 7.- Studying the effect of the exposure to light on the expression of different genes involved in the pro- and anti-apoptotic pathways. 8.- Determining whether the light causes any changes in the expression of some calciumlinking proteins, present in the retinal cells, and the eventual prevention of this effect implanting intraocular lenses which suppress the blue and violet part of the luminous spectrum. 9.- Studying the expression changes in Trk-B, the BDNF receptor, as a consequence of the exposure to the light Assisting to the knowledge of the metaloprotease regulation in the pathogenesis of the light-induced retinal degeneration Evaluating the effect of the exposure to the light on the expression of genes TIMP-1 and TIMP-2, and analizing the eventual prevention of this effect by means of selectiveabsorption filters for short wavelengths. 3 HYPOTHESIS 3.1 Conceptual hypothesis: Short wavelength light causes a neurodegenerative process in the visual system, which can be reduced using optic filters to absorb that radiation. 3.2 Physiological hypothesis: The optic filters with a selective absorption of the visible spectrum, which contain yellow pigments, protect the retina. 4 METHODS Pigment rabbits aged between years old from Universidad Complutense de Madrid were used in this study. Sample was divided in two groups according the intraocular lens implanted: Control groups is formed by rabbits without cataract surgery, study group comprised rabbits implanted with a blue light absorbing intraocular lens in the right eye and implanted with a clear intraocular lens in the fellow eye. Rabbits were exposed to white light, white light in absence of blue light (named yellow light) or blue light. A group of rabbits were not exposed to light. 3

4 Sample distribution is shown in the following table. Study (catarat surgery) Light Control (no surgery) RE Y-IOL LE C-IOL Start End Start End No exposed White light Yellow light Blue light TOTAL Table 1. Sample distribution Intraocular lens used in the surgeries were Acrysoft (clear intraocular lens) and Acrysoft Natural (yellow intraocular lens). In the following figure are presented the spectral characteristics of the intraocular lenses in comparison with a natural crystalline lens. Figure 1. Spectral characteristics of the intraocular lens used in this study Light sorces were located in the animals cage to obtain a controlled and homogeny light. Yellow light was obtained with a white fluorescent tube covered by a blue light absorbing filter E-Colour #010 (ROSCO Ibérica S.A.): Medium Yellow (Transmission = 83,91%). Light intensity was calculated according the normal exposure of a human eye. Rabbits were exposed to 12hours light / 12 hours darkness cycles for 2 years. Following the irradiation period, rabbits were killed and the eyes were maintained on formaldehyde. Following the normal treatment process, eyes were analyzed. It was realized a structural, genetic and inmunohistological analysis. 5 RESULTS 5.1 Gene-expression analysis Expression of pro-apoptotic and anti-apoptotic genes Basically, the exposure to different types of light, in circadian cycles, for long periods of time, does not module the expression of those genes related to the cell-death processes by apoptosis. As far as Bad (pro-apoptotic) is concerned, the continuous exposure to white light increases its expression 2.5 times, to blue light 6.8 times, and to yellow light 3.1 times. 4

5 The intraocular lenses can take the expression of this gene to levels near those of white light. Figure 2. Expression of gene Bad in the retina of rabbits exposed to lighting for 2 years. NE: non-exposed; B: white light; Az: blue light; Am: white light without the blue part of the spectrum. LIT: clear intraocular lens; LIA: yellow intraocular lens. The expression levels of caspase-1 do not change in any of the experimental groups, or implanting intraocular yellow lenses. On the other hand, the gene Bax (proapoptotic) does not have any changes in the analized experimental conditions. Figure 3. Expression of the caspase-1 gene in the retina of rabbits exposed to lighting for 2 years. NE: non exposed; B: White light; Az: Blue light; Am: White light without the blue part of the spectrum. LIT: clear intraocular lens; LIA: yellow intraocular lens. Figure 4. Expression of the gene Bax in the retina of rabbits exposed to lighting for 2 years. NE: nonexposed; B: white light; Az: blue light; Am: white light without the blue part of the spectrum. LIT: clear intraocular lens; LIA: yellow intraocular lens Out of these data, it is clear that, in the mechanisms of long-term light-induce dcell death by apoptosis in circadian cycles, the main signaling pathway includes Bad. 5

6 Regarding the antiapoptotic genes, our results show that there are no expression changes in Bcl-2 under any of the experimental scenaries, while the Bcl-XL (antiapoptotic) levels are increased in the animals implanted with a yellow intraocular lens. Figure 5. Expression of the genes. Bcl_XL and Bcl-2 in the retina of rabbits exposed to lighting for 2 years. NE: no-exposed; B: white light; Az: blue light; Am: white light without the blue part of the spectrum. LIT: clear intraocular lens; LIA: yellow intraocular lens. In the lab animals used, the expression of c-fos is increased by 3.3 times in the animals exposed to blue light, and 2.7 times in those exposed to white, blue-free light; however, the expression levels go down to 1.6 times in the animals wearing a yellow intraocular lens. With regards to the expression of d-jun, it is not modified by the different types of light, but it is very low (- 2.2 times less) in the animals with the yellow intraocular lens. 6

7 Figure 6. Expression of genes c-fos, and d-jun in the retina of rabbits exposed to lighting for 2 years. NE: non-exposed; B: white light; Az: blue light; Am: white, blue-free light. LIT: clear intraocular lens; LIA: yellow intraocular lens. 2+-linking proteins Expression of genes related to the Ca The results achieved in this study show that the expression of calbindine is not modified as a consequence of exposure to light, or implanting yellow intraocular lenses; the protein S100a6 stays at basal levels, with no changes in any of the experimental arranged situations. Figure 7. Expression of the genes calbindine, S100a6 in the retina of rabbits exposed to lighting for 2 years. NE: non-exposed; B: white light; Az: blue light; Am: white light without the blue part of the spectrum. LIT: clear intraocular lens; LIA: yellow intraocular lens. Finally, calretinine shows lower expression levels in the animals submitted to white, bluefree light (-1.3 times), and in the animals wearing a yellow intraocular lens. Figure 8. Expression of the calretinine gene in the retina of rabbits exposed to lighting for 2 years. NE: non-exposed; B: white light; Az: blue light; Am: white light without the blue part of the spectrum. LIT: clear intraocular lens; LIA: yellow intraocular lens. 7

8 It results very attractive the calcium hypothesis in the cell degenerative mechanisms, especially in the neurons. However, given the complexity of the regulating mechanisms, the available data do not lead to any definite conclusions Expression of the gene for the neurotrophin receptor Trk-B In this work, we saw that long-term exposure to light regulates up the expression of TDK-B: 1.8 times the white light, 2.3 the blue light, 3.9 the yellow light; in the animals wearing an intraocular lens, the increase in the expression of Trk-B was 4.2 times with regards to the basal values. Figure 9. Expression of the gene Trk-B in the retina of rabbits exposed to permanent lighting for 2 years. NE: non-exposed; B: white light; Az: blue light; Am: white, blue-free light. LIT: clear intraocular lens; LIA: yellow intraocular lens Expression of genes realted to some metaloproteases The results of this work show that light, or the intraocular implantation of a yellow lens, do not modify the expression of MMP-2. Figure 10. Expression of the genes MMP-2, MMP-3, and MMP-9 in the retina of rabbits exposed to lighting for 2 years. NE: non-exposed; B: white light; Az: blue light; Am: white light without the blue part of the spectrum. LIT: clear intraocular lens; LIA: yellow intraocular lens On the contrary, in the animals exposed to blue-free light, and in the animals wearing a yellow intraocular lens, the expression of MMP-3 is increased 2.9 and 3.6 times, respectively. And a nearly identical evolution can be seen for MMP-9, also regulated up in the animals exposed to blue light (3.1 times), to white, blue-free light (4.6 times), and implanted with a yellow intraocular lens (4.2 times). 8

9 Figure 11. Expression of genes MMP-2, MMP-3, and MMP-9 in the retina of rabbits exposed to lighting for 2 years. NE: non-exposed; B: white light; Az: blue light; Am: white light without the blue part of the spectrum. LIT: clear intraocular lens; LIA: yellow intraocular lens Expression of TIMPs genes The genes encoding for the specific tisular inhibitors of MMPs, TIMP1 and TIMP2, are normally expressed in hte rabbit retina, and its expression is not modified in white-light invironments after implanting the intraocular lens. However, the yellow-light environment involves a very significant fall in the expression of TIMP1, obvious but non-quantified in comparison with the white-light environment. In a yellow-light environment, the intraocular lens does not affect the expression pattern of TIMP1. However, the exposure to blue light increases the expression of TIMP1, similar to the case of exposure to White light in animals wearing a yellow intraocular lens but 3 times higher in absence of htthat protection. That is, in blue-light environments the yellow intraocular lens avoids the rise in the expression levels of TIMP1. Figure 12. Results of the gene expression of TIMP-1 Regarding TIMP2, the expression levels were very similar in the groups of animals exposed to environments of white and yellow light, and the implantation of a yellow intraocular lens did not modify the expression of this gene. However, the exposure to blue light increases the expression of this gene, what can be stopped by the yellow intraocular lens; in this group of animals, those without the lens showed expression levels 6 times higher. 9

10 Figure 13. Results of the gene expression of TIMP-2 6 CONCLUSIONS 1.- The chronic exposure to circadian lighting, with different types of light, causes a fall down in the cell density of the inner and outer nuclear layers, as well as in the ganglionar layer. 2.- The greatest loss of retinal cells takes place in the animals submitted to blue light. This deficit can be partially avoided filtering the short wavelengths. 3.- The cell loss in the outer and inner nuclear layers is caused by apoptosis. 4.- Rods were the photoreceptors most severely affected by their exposure to short wavelengths. This harmful effect is partially corrected filtering those bands. 5.- The retinal cell connectivity is less in those animals submitted to circadian lighting. The deffect is greater in the animals submitted to blue light. These effects can be partially corrected with the yellow optic filters. 6.- Exposure to short-wavelength light favors apoptosis, as there is an increase in the expression of the pro-apoptotic gene Bad, and a reduction in the expression of the antiapoptotic gene Bcl-XL. The harmful blue-light effects can be prevented absorbing these short wavelengths with selective optic filters. 7.- The filtering of the short wavelengths inhibits the start signaling for apoptosis, as it modifies the expression of the protooncogenes c-fos and d-jun. 8.- The filtering of the short wavelengths causes an up-expression of the gene Trk-B. This can be a response to the neuroprotective stimuli caused by this molecule. 9.- The chronic exposure to circadian lighting increases the expression of some MMPs involved in the onset of drusen. It is unknown if this increase favors their formation, or prevents the accumulation of these deposits The exposure to blue light causes an increase in the expression of TIMP-1 and TIMP-2, which comes down using selective short-wavelength optic filters The absorption of the short wavelengths using a selective optic filter is beneficial, and causes the same results irrespective their application on the very light source, or in the implanted intraocular lens. In other words, the short-wavelength filtering effects take place irrespective the filter placing with regards to the retina. 10