B-wave S13

[Ermengardi Atkisson]

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Considering the increasing evidence of a rapid myopic trend in the younger cohorts of the global population, regulation of myopia progression has become a priority for the scientific community, eye care practitioners and policy makers. Different optical strategies have been developed and implemented over the past 10 to 15 years in an attempt to address this evolving global health concern1. Beyond the pattern of image formation, it is increasingly evident that the spectral composition and intensity of light are important to consider when attempting to interfere with myopia progression2,3. The contribution of intrinsically photosensitive retinal ganglion cells (ipRGCs) to different physiological functions has been elucidated in recent years and it is now possible to link ipRGCs to the modulation of the retinal activity4. Dopamine (DA) has been proposed as one of the neurotransmitters involved in the control of several physiological processes, including eye growth and refractive error development5. After the early discoveries of Stone et al. relating a decline in retinal dopamine with deprivation myopia, the potential involvement of DA in myopia development has been the subject of extensive review6. A previous study using electrophysiological techniques suggests that dopaminergic neurons receive excitatory input from synapses with ON bipolar cells in the inner plexiform layer (IPL)7.

Dopaminergic amacrine cells (DACs) are the main source of retinal dopamine, and are activated by rods, cones, and ipRGCs in response to light8. The activation of DA receptors, expressed by photoreceptors and amacrine cells, regulates gap junctions between different retinal elements and affects the amplitude of the electroretinogram (ERG) b-wave9. In fact, DACs can also be upregulated by stimulation of the ON bipolar circuit10. ON bipolar cells provide excitatory synaptic input to DACs, triggering dopamine release and the regulation of light responses in the inner retina. Li et al. injected chicken eyes with 6-OHDA which depletes DA from DAC. Authors showed small changes in dopaminergic pathways as measured with ERGs and oscillatory potentials. However, such changes were apparently strong enough to block development of deprivation myopia11. This is confirmed by the changes observed in the a- and b-waves of ERGs and oscillatory potentials12.

In clinical and experimental studies of human retinal function, the ERG b-wave is a measure that is commonly evaluated in research19. An important component of the ERG curve, the b-wave primarily reflects the post-synaptic retinal cells of the photoreceptors, namely the bipolar cells. Blocking the neurotransmission from photoreceptors to bipolar cells eliminates the b-wave20,21. In photopic ERG recordings, the b-wave is shaped by depolarizing ON-bipolar cells in the ascending phase and hyperpolarizing OFF-bipolar and horizontal cells in the descending phase, which pull the depolarization towards baseline19. The general function of retinal ganglion cells can be assessed using PERG recordings, where P50 and N95 peaks are of particular interest to researchers. In transient PERG, ON and OFF pathways contribute equally to the waveform. However, while P50 originates from both firing and non-firing of the pathways, N95 is suggested to be mainly related with firing activity22.

Melanopsin-expressing ipRGCs are a potential target system for a physiological enhancement of DA levels in the myopic eye, as they have been shown to project via their axon collaterals onto DACs23 and thereby modulate DA levels in response to light24,25.

The axons of ipRGCs pass through the optic nerve head, which corresponds to the blind-spot, and express melanopsin, as shown in rodents and humans26,27,28. Therefore, stimulation of the blind-spot with blue light overlapping with the sensitivity of melanopsin (around 480 nm29) could generate a retrograde effect of upregulation of DA secretion by DACs in the IPL30. Synaptic contact with bipolar cells at the same level might also be observed. This effect can be indirectly evaluated by analyzing the b-wave31.

Therefore, it was hypothesized that direct stimulation of the blind-spot with short-wavelength light would induce a retrograde effect on retinal ganglion cells, measurable by PERG, and that their interaction with bipolar cells in the IPL could be evaluated through the b-wave of full-field ERG (ffERG). We further hypothesized that such changes would be stronger in the myopic retina where a lack of DA could make them more sensitive to DA released in response to short-wavelength light32, and thus result in changes to the ERG response following blue light stimulation.

To the best of our knowledge, the present study is the first evaluating the effect of stimulating the blind-spot with blue light using a virtual reality (VR) system on the electrophysiological response of the human retina. The results of this study have revealed two important points. First, that the level of retinal activity measured by photopic 3.0 of ffERG and PERG, potentially related to the dopaminergic path, can be increased in myopic eyes after blue light stimulation of the blind-spot. This may reflect a retrograde feedback effect of the ipRGCs at the level of the IPL where they connect with amacrine and bipolar cells as shown by the PERG and b-wave response. Second, the effect we have shown was observed in myopes and not in non-myopes, and therefore this stimulation technology may have major implications for future myopia control.

Although statistical analyses did not indicate a significant change in the implicit time following blind-spot stimulation, the small, but apparent, acceleration of the P50 peak should not be overlooked as it suggests that blue light stimulation may induce a faster response of ON and OFF pathways. In fact, these non-significant trends in implicit time are worth investigating in future studies with a greater number of participants and can be used as informative average of expected values for similar studies.

Myopic eyes have been shown to have lower levels of dopaminergic activity within the retina and susceptibility to form deprivation myopia in animal models was higher when the DOPAC/ DA ratio was lower33. This suggests that lower metabolic activity involving DA is associated with a stronger predisposition to develop higher degrees of myopia33.

Based on the simultaneous upregulation of the PERG and b-wave observed in the present study in myopic eyes, we can advance with a proposed mechanism that confirms the hypothesis raised in this study. Connectivity between ipRGCs and DACs has been established at the level of the IPL34. Zhou et al. have proposed a mechanism in which bright flickering light that stimulates the ON pathway and ipRGCs can alter DA synthesis and release6. In the present study a flickering blue light within the maximum range of sensitivity of melanopsin-containing ipRGCs has been used. A relationship between ipRGCs and DACs has been established as both are driven by ON bipolar cells stratifying in the outermost IPL10. Therefore, it is plausible that the activation of ipRGCs, as measured by the PERG, could be responsible for the observed b-wave changes. Such changes may reflect the upregulation of DA as a result of the link between ipRGCs and DACs at the level of the IPL. The protective role of the activation of the ON channel in myopia has been documented in several animal models35. The present results open the door to the use of this intervention approach in humans.

Additional research has found that exposure to bright light during outdoor activities is effective at delaying the onset of myopia and that this effect may be related to higher DA activity36,37. Therefore, the results of the current study point to the potential of blind-spot stimulation with short-wavelength light to elicit a retrograde effect in the IPL. This retrograde effect may be reflected in the increased PERG amplitude after blind-spot stimulation that is mirrored in the b-wave amplitude of myopes, independent of the degree of change created by the blue light stimulus at each retinal layer. Similar b-wave behavior for different levels of change in PERG activity could indicate a binary gate. In such a case the increase in b-wave activity does not increase linearly with the degree of PERG amplitude change. Rather, the b-wave increases to a similar level irrespective of the PERG response once the PERG is upregulated. This is true even at different levels across individuals, as the b-wave upregulation is similar for different participants.

In this study the blind-spot, which corresponds to the optic nerve head, was stimulated with blue-light via VR system. However, the optic nerve head can be stimulated also via silent substitution technology44. Eye movements could be recorded during stimulation in future experiments as soon as the technical limitations of an eye-tracker in the VR system are solved and allow a precise fixation determination.

Another potential limitation of the present study is that measurements were not taken at the same time of the day for all participants. However, for each participant, PERG and b-wave recordings were obtained within the same period of the day in order to avoid the effect of circadian changes when comparing the retinal response4. We conducted a brief analysis to verify whether participants measured during the morning differed from those measured during the afternoon (data not shown in this study). Despite differences in absolute values, an increase in retinal activity after 20 min was observed independently of the time of the day.

The assumption that IPL is potentially mediated by DA release after blind-spot stimulation is consistent with the structure of the ON and OFF pathway circuits in the mammalian retina at the level of the IPL and could be mediated by ON bipolar cells synapsing with ipRGCs and amacrine cells4. Therefore, based on the present results we can hypothesize that the upregulation of retinal electrical activity could be related to an upregulation of DA release. Interestingly, this effect is quite selective of myopic eyes, rather than emmetropic eyes, which showed consistent activity before and after stimulation. This suggests that the blue light stimulus only produced an improvement in myopes, where retinal activity may be compromised due to some anatomical or physiological changes, while normal eyes did not experience any effect of the treatment. In a similar way, previous studies reported improvements in the retinal activity of diabetic animals after administrating treatment, while in normal eyes no influence of the treatment was observed52,53,54.

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