Implication of Melanopsin and Trigeminal Neural Pathways in Blue Light Photosensitivity in vivo

Abstract

Photophobia may arise from various causes and frequently accompanies numerous ocular diseases. In modern highly illuminated world, complaints about greater photosensitivity to blue light increasingly appear. However, the pathophysiology of photophobia is still debated. In the present work, we investigated in vivo the role of various neural pathways potentially implicated in blue-light aversion. Moreover, we studied the light-induced neuroinflammatory processes on the ocular surface and in the trigeminal pathways. Adult male C57BL/6J mice were exposed either to blue (400-500 nm) or to yellow (530-710 nm) LED light (3 h, 6 mW/cm²). Photosensitivity was measured as the time spent in dark or illuminated parts of the cage. Pharmacological treatments were applied: topical instillation of atropine, pilocarpine or oxybuprocaine, intravitreal injection of lidocaine, norepinephrine or "blocker" of the visual photoreceptor transmission, and intraperitoneal injection of a melanopsin antagonist. Clinical evaluations (ocular surface state, corneal mechanical sensitivity and tear quantity) were performed directly after exposure to light and after 3 days of recovery in standard light conditions. Trigeminal ganglia (TGs), brainstems and retinas were dissected out and conditioned for analyses. Mice demonstrated strong aversion to blue but not to yellow light. The only drug that significantly decreased the blue-light aversion was the intraperitoneally injected melanopsin antagonist. After blue-light exposure, dry-eye-related inflammatory signs were observed, notably after 3 days of recovery. In the retina, we observed the increased immunoreactivity for GFAP, ATF3, and Iba1; these data were corroborated by RT-qPCR. Moreover, retinal visual and non-visual photopigments distribution was altered. In the trigeminal pathway, we detected the increased mRNA expression of cFOS and ATF3 as well as alterations in cytokines' levels. Thus, the wavelength-dependent light aversion was mainly mediated by melanopsin-containing cells, most likely in the retina. Other potential pathways of light reception were also discussed. The phototoxic message was transmitted to the trigeminal system, inducing both inflammation at the ocular surface and stress in the retina. Further investigations of retina-TG connections are needed.

Keywords: blue light; dry eye; melanopsin; neurotoxicity; ocular nociception; photophobia; trigeminal pathway.

FIGURE 1

Custom-mounted illumination system. (A) Illumination system and relative spectra of LED sources. (B) When placed together (and not in separate compartments), mice exposed to blue illumination demonstrated light aversion by hiding behind each other; such behavior did not take place for yellow exposure. (C) Behavior test: mice are placed in half-illuminated boxes and allowed to move freely. As in the previous figure, mice exposed to blue illumination demonstrated a strong light aversion, as compared to the yellow one under which animals preferred to stay.

FIGURE 2

Clinical assessments. Measurements were made at three time points: before hν, before the beginning of illumination; after hν, directly after 3 h of illumination; recovery 3d, after 3 days of recovery in standard illumination conditions of animal unit. (A) Measurement of corneal mechanical sensitivity performed by means of von Frey hair test. Greater values mean lower corneal sensitivity. Statistical significance: blue illumination group: before hν vs. after hν – q = 0.0361, p = 0.1031; before hν vs. recovery 3d – q = 0.0003, p = 0.003; after hν vs. recovery 3d – q = 0.0136, p = 0.0260; recovery 3d group: blue vs. yellow – q = 0.0041, p = 0.0020. (B) Measurement of tear quantity performed by means of phenol red thread test placed into the eye for 30 s. Greater distances mean more important lacrimation. Statistical significance for the blue illumination group: before hν vs. after hν – q = 0.0403, p = 0.0192; before hν vs. recovery 3d – q = 0.0498, p = 0.0475. Blue and yellow bars correspond to blue and yellow exposures, respectively. All the data are presented as mean ± SEM. Differences were considered significant when p < 0.05 (^∗/^∧), p < 0.01 (^∗∗/^∧∧), p < 0.001 (^∗∗∗/^∧∧∧) or p < 0.0001 (^∗∗∗∗/^∧∧∧∧). Stars correspond to comparisons between values at different time points, within one spectrum. Carets correspond to comparison between blue-illuminated and yellow-illuminated mice, at the same time point. Red color means increase and blue color decrease in values.

FIGURE 3

IVCM results. Representative images of non-invasive IVCM examination performed directly after exposure to light (A, no recovery) or after 3 days of recovery in standard illumination conditions of animal unit (B, recovery). Alterations were observed in the three following corneal layers: superficial epithelium (cell nuclei in blue-illuminated mice became more hyperreflective), sub-basal plexus (dendritic cells are marked by circles) and stroma (activated keratocytes are marked by arrows).

FIGURE 4

Light-induced retinal inflammation (1). Immunohistochemistry was performed on the retinas of blue- (b) and yellow-exposed (y) mice either immediately after illumination (nRec) or in 3 days of recovery (Rec). Results of anti-GFAP (A) and anti-ATF3 (B, immuno-activated cells are marked by arrowheads) stainings are presented. Magnification is 10×(A,B), scale bars correspond to 100 μm.

FIGURE 5

Light-induced retinal inflammation (2). (A) Immunohistochemistry was performed on the retinas of blue- (b) and yellow-exposed (y) mice either immediately after illumination (nRec) or in 3 days of recovery (Rec). Results of anti-Iba1 staining is presented. Magnification is 20x, scale bar corresponds to 100 μm. (B–D) Results of RT-qPCR analysis on the retinas: mRNA expression of GFAP (B), ATF3 (C) and Iba1 (D). Statistical significance: - GFAP: blue no recovery vs. recovery – q = 0.0101, p = 0.0096; recovery blue vs. yellow – q = 0.0074, p = 0.0070; - ATF3: blue no recovery vs. recovery – q = 0.0230, p = 0.0219; no recovery blue vs. yellow – q = 0.0057, p = 0.0054; - Iba1: blue no recovery vs. recovery – q = 0.0118, p = 0.0113; yellow no recovery vs. recovery – q = 0.0428, p = 0.0814. Blue and yellow bars correspond to blue and yellow exposures, respectively; clear and hatched bars correspond to the time points of mice dissection, either directly after illumination (no recovery) or in 3 days of recovery (recovery), respectively. All the data are presented as mean ± SEM. Differences were considered significant when p < 0.05 (^∗/^∧), p < 0.01 (^∗∗/^∧∧), p < 0.001 (^∗∗∗/^∧∧∧) or p < 0.0001 (^∗∗∗∗/^∧∧∧∧). Stars correspond to comparisons between blue-illuminated and yellow-illuminated mice, within one recovery or non-recovery group. Carets correspond to comparison of mice assessed directly after illumination to the ones assessed after 3 days of recovery, within the same spectra. Red color means increase and blue color decrease in values.

FIGURE 6

Retina-related behavioral tests. Graphs illustrate the time spent in the illuminated part of the box during the chosen representative periods. For more detail, see “Materials and Methods” section. (A) Pupils were dilated with atropine (atro) or constricted with pilocarpine (pilo). One drop per eye was instilled (inst) bilaterally 5 min before the start of light exposure (1st hour: PBS blue vs. yellow – q = 0.0004, p = 0.0003; pilo blue vs. yellow – q = 0.0057, p = 0.0108; yellow PBS vs. atro – q < 0.0001, p < 0.0001; 3 h: PBS blue vs. yellow – q < 0.0001, p < 0.0001; pilo blue vs. yellow – q = 0.0006, p = 0.0012; yellow PBS vs. atro – q < 0.0001, p < 0.0001). (B) Visual receptors’ pathway was blocked (VR blocker). 2 μL of drug (the composition is described in “Materials and Methods” section) was injected intravitreally (ivt) bilaterally 5 min before the start of light exposure (1st hour: PBS blue vs. yellow – q = 0.0006, p = 0.0012; VR blocker blue vs. yellow – q = 0.0090, p = 0.0086; 3 h: PBS blue vs. yellow – q < 0.0001, p < 0.0001; VR blocker blue vs. yellow – q = 0.0003, p = 0.0002). (C) Melanopsin antagonist was injected (opn4 antago) intraperitoneally (ip, 30 mg/kg) 15 min before the start of light exposure (1st hour: blue DMSO vs. opn4 antago – q = 0.0223, p = 0.0212; DMSO blue vs. yellow – q = 0.0123, p = 0.0117; 3 h: blue DMSO vs. opn4 antago – q = 0.0155, p = 0.0147; DMSO blue vs. yellow – q = 0.0128, p = 0.0122). Blue and yellow bars correspond to blue and yellow exposures, respectively; clear bars and hatched bars correspond to animals with control (vehicle – PBS or DMSO) or specific drug treatments, respectively. All the data are presented as mean ± SEM. Differences were considered significant when p < 0.05 (^∗/^∧), p < 0.01 (^∗∗/^∧∧), p < 0.001 (^∗∗∗/^∧∧∧) or p < 0.0001 (^∗∗∗∗/^∧∧∧∧). Stars correspond to comparisons between blue-illuminated and yellow-illuminated mice, treated with the same drug. Carets correspond to comparisons between control and drug-treated animals. Red color means increase and blue color decrease in values. For the results close to be significant, correspondent p- and q-values are marked on the graph.

FIGURE 7

Role of retinal photoreceptors (1). Immunohistochemistry was performed on the retinas of blue- (b) and yellow-exposed (y) mice either immediately after illumination (nRec) or in 3 days of recovery (Rec). Results of anti-Cone Arrestin (A) and anti-opn4 (B) stainings are presented. Insets with higher zoom are provided. Magnification is 20×(A) and 10×(B), scale bars correspond to 100 μm.

FIGURE 8

Role of retinal photoreceptors (2). (A) Immunohistochemistry was performed on the retinas of blue- (b) and yellow-exposed (y) mice either immediately after illumination (nRec) or in 3 days of recovery (Rec). Results of anti-opn5 (C, localization of neuropsin-expressing cells is circled) staining is presented. Magnification is 20×, scale bars corresponds to 100 μm. (B,C) Results of RT-qPCR analysis on the retinas: mRNA expression of opn4 (B; blue no recovery vs. recovery – q = 0.0174, p = 0.0166; yellow no recovery vs. recovery – q = 0.0499, p = 0.0951) and opn5 (C). Blue and yellow bars correspond to blue and yellow exposures, respectively; clear and hatched bars correspond to the time points of dissection, either directly after illumination (no recovery) or in 3 days of recovery (recovery), respectively. All data are presented as mean ± SEM. Differences were considered significant when p < 0.05 (^∗/^∧), p < 0.01 (^∗∗/^∧∧), p < 0.001 (^∗∗∗/^∧∧∧) or p < 0.0001 (^∗∗∗∗/^∧∧∧∧). Carets correspond to comparison of mice assessed directly after illumination to the ones assessed after 3 days of recovery, within the same spectra. Red color means increase and blue color decrease in values.

FIGURE 9

Trigeminal pathway-related behavioral tests. Graphs illustrate the time spent in the illuminated part of the box during the chosen representative periods. For more detail, see “Materials and Methods” section. (A) Ocular surface was anesthetized with oxybuprocaine (oxybu). One drop per eye was instilled (inst) bilaterally directly before the start of light exposure (1st hour: PBS blue vs. yellow – q = 0.0008, p = 0.0008; oxybu blue vs. yellow – q < 0.0001, p < 0.0001; 3 h: PBS blue vs. yellow – q < 0.0001, p < 0.0001; oxybu blue vs. yellow – q = 0.0001, p = 0.0001). (B) Intraocular trigeminal afferents were anesthetized with lidocaine (lido). 2 μL of drug was injected intravitreally (ivt) bilaterally 5 min before the start of light exposure (1st hour: PBS blue vs. yellow – q = 0.0007, p = 0.0006; 3 h: yellow PBS vs. lido – q = 0.0210, p = 0.0200; PBS blue vs. yellow – q = 0.0004, p = 0.0003). (C) Intraocular blood vessels were constricted with norepinephrine (norip). 2 μL of drug was injected intravitreally (ivt) bilaterally 5 min before the start of light exposure (1st hour: PBS blue vs. yellow – q = 0.0003, p = 0.0003; norip blue vs. yellow – q < 0.0001, p < 0.0001; 3 h: PBS blue vs. yellow – q < 0.0001, p < 0.0001; norip blue vs. yellow – q < 0.0001, p < 0.0001). Blue and yellow bars correspond to blue and yellow exposures, respectively; clear bars and hatched bars correspond to animals with control (vehicle – PBS) or specific drug treatments, respectively. All the data are presented as mean ± SEM. Differences were considered significant when p < 0.05 (^∗/^∧), p < 0.01 (^∗∗/^∧∧), p < 0.001 (^∗∗∗/^∧∧∧) or p < 0.0001 (^∗∗∗∗/^∧∧∧∧). Stars correspond to comparisons between blue-illuminated and yellow-illuminated mice, treated with the same drug. Carets correspond to comparisons between control and drug-treated animals. Red color means increase and blue color decrease in values. For the results close to be significant, correspondent p- and q-values are marked on the graph.

FIGURE 10

Phototoxicity marks in the trigeminal pathway. (A–C) Results of RT-qPCR analysis on the TGs: mRNA expression of cFOS (A), Iba1 (B), and ATF3 (C). Statistical significance: cFOS: blue no recovery vs. recovery – q = 0.0129, p = 0.0061; yellow no recovery vs. recovery – q = 0.0339, p = 0.0323; ATF3: blue no recovery vs. recovery – q = 0.0076, p = 0.0072; recovery blue vs. yellow – q = 0.0243, p = 0.0231. (D–F) Results of RT-qPCR analysis on the brainstems: mRNA expression of cFOS (D; blue no recovery vs. recovery – q < 0.0001, p < 0.0001; yellow no recovery vs. recovery – q < 0.0001, p < 0.0001), Iba1 (E) and ATF3 (F). Blue and yellow bars correspond to blue and yellow exposures, respectively; clear and hatched bars correspond to the time points of mice dissection, either directly after illumination (no recovery) or in 3 days of recovery (recovery), respectively. All the data are presented as mean ± SEM. Differences were considered significant when p < 0.05 (^∗/^∧), p < 0.01 (^∗∗/^∧∧), p < 0.001 (^∗∗∗/^∧∧∧) or p < 0.0001 (^∗∗∗∗/^∧∧∧∧). Stars correspond to comparisons between blue-illuminated and yellow-illuminated mice, treated with the same drug. Carets correspond to comparison of mice assessed directly after illumination to the ones after 3 days of recovery, within the same spectra. Red color means increase and blue color decrease in values.

FIGURE 11

Cytokine profile in trigeminal pathways. Results of mRNA expression of TGFβ2 and TNFα on the TGs (A,B) and brainstems (C,D). Statistical significance for the TG: TGFβ2 blue no recovery vs. recovery – q = 0.0187, p = 0.0178. Statistical significance for the brainstem; TGFβ2: blue no recovery vs. recovery – q = 0.0169, p = 0.0161; yellow no recovery vs. recovery – q = 0.0041, p = 0.0020; TNFα: blue no recovery vs. recovery – q = 0.0197, p = 0.0188; recovery blue vs. yellow – q = 0.0005, p = 0.0005. Blue and yellow bars correspond to blue and yellow exposures, respectively; clear and hatched bars correspond to the time points of dissection, either directly after illumination (no recovery) or in 3 days of recovery (recovery), respectively. All the data are presented as mean ± SEM. Differences were considered significant when p < 0.05 (^∗/^∧), p < 0.01 (^∗∗/^∧∧), p < 0.001 (^∗∗∗/^∧∧∧) or p < 0.0001 (^∗∗∗∗/^∧∧∧∧). Stars correspond to comparisons between blue-illuminated and yellow-illuminated mice, treated with the same drug. Carets correspond to comparison of mice assessed directly after illumination to the ones assessed after 3 days of recovery, within the same spectra. Red color means increase and blue color decrease in values.

FIGURE 12

Role of melanopsin in corneal sensitivity. Measurement of corneal mechanical sensitivity performed by means of von Frey test. Greater values mean lower corneal sensitivity. Test was performed in naïve mice (clear bars, the same results as the ones presented in the Figure 2) and in mice intraperitoneally (ip, 30 mg/kg) injected with melanopsin antagonist (opn4 antago) 15 min before the start of the test. For more detail, see “Materials and Methods” section. Statistical significance for the recovery 3d group: blue naïve vs. yellow naïve – q = 0.0010, p = 0.0010, blue naïve vs. blue antago opn4 – q = 0.0008, p = 0.0005, blue naïve vs. yellow antago opn4 – q = 0.0004, p = 0.0001. Measurements were made at three time points: before hν, before the beginning of illumination; after hν, directly after 3 h of illumination; recovery 3d, after 3 days of recovery in standard illumination conditions of animal unit. Blue and yellow bars correspond to blue and yellow exposures, respectively. All the data are presented as mean ± SEM. Differences were considered significant when p < 0.05 (^∧), p < 0.01 (^∧∧), p < 0.001 (^∧∧∧) or p < 0.0001 (^∧∧∧∧).

FIGURE 13

Model of blue-phototoxicity. Proposed scheme of blue light provoked time-dependent immune and inflammatory responses in the ocular surface, trigeminal pathways and the retina. For more detail, see the “Discussion” section.