Decreased Memory and Learning Ability Mediated by Bmal1/M1 Macrophages/Angptl2/Inflammatory Cytokine Pathway in Mice Exposed to Long-Term Blue Light Irradiation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is an interesting and informative manuscript. My comments are minor:

General comments:

-       I am always cautious about interpreting animal behavioural studies in the context of humans.

 

-       Was the timing of the daily irradiation period consistent throughout the trial period? What was the window?

 

-       What is the rational for an irradiation period of 10 minutes?

 

-       Murine animals, including mice and rats, are dichromats. As a result, their color vision is limited compared to humans, who are typically trichromats. The exact range of colors that murine animals can see is not fully understood, but research suggests that they are most sensitive to wavelengths in the ultraviolet (UV) and short-wavelength visible light range (blue to green). They have limited sensitivity to longer wavelengths (yellow, orange, and red). This may account for the different responses to higher wavelengths of light in this study and others.

Figure 1: Spectral sensitivity function of the cone, rod, and ipRGC 

(Jacobs, G. H., Neitz, J., & Deegan, J. F. (1991). Retinal receptors in rodents maximally sensitive to ultraviolet light. Nature, 353(6345), 655-656).

-       Line 72: Does the light emitted by the fluorescent lamps contain blue light? If yes, how would we overcome this confounding factor?

 

-       Line 193-194: Were other factors that impact cytokine levels in the brain, or conditions that cause neuro-inflammation, taken into account when measuring the cytokine levels?

 

-       Line 212: Ag-1 should be corrected to Arg-1

 

-       Line 232: Could you please clarify the distinction between M1 and M2 macrophages in your study? It's generally understood that M2 macrophages have anti-inflammatory properties, while inflammatory cytokines such as TNF-alpha and IL-6 are usually secreted by M1 macrophages. Clarifying this distinction would help in better understanding the role of macrophages in your findings.

 

-       Line 219: ?Mnal1 -> Bmal1

 

-       Line 237: “Microglia shift to M2 macrophages and directly or indirectly induce the secretion of inflammatory cytokines…:

 

 

Minor syntax errors throughout.

 

Comments on the Quality of English Language

The English is good.

Author Response

Response to the Reviewers’ comments

 

To Reviewer 1

Thank you for your detailed and valuable comments. We have carefully considered the reviewer's point of view and addressed each as follows.

 

1. Was the timing of the daily irradiation period consistent throughout the trial period? What was the window?

Response: Thank you for your comment. Irradiation was performed at 10:00 a.m. throughout the test period. The windows were equipped with an anti-reflection coating (WBMA-15CO2-10-lR1; Sigmakoki Co. Ltd., Tokyo, Japan).

Line 77-78

Irradiation was performed at a constant time, 10:00 a.m., during the test period.

 

2. What is the rational for an irradiation period of 10 minutes?

Response: Thank you for your query. Irradiation was performed for 10 minutes to ensure an irradiation energy amount of 40 kJ/m². This was based on our previous blue light irradiation experiments, which revealed that irradiation of 40 kJ/m²can impact living organisms. (Reference 10)

 

3. Murine animals, including mice and rats, are dichromats. As a result, their color vision is limited compared to humans, who are typically trichromats. The exact range of colors that murine animals can see is not fully understood, but research suggests that they are most sensitive to wavelengths in the ultraviolet (UV) and short-wavelength visible light range (blue to green). They have limited sensitivity to longer wavelengths (yellow, orange, and red). This may account for the different responses to higher wavelengths of light in this study and others.

Response: Thank you for your important comments. As stated, mice are most sensitive to UV and blue-to-green light; hence, exposure to UV and blue-to-green light may exert a stronger response to learning and memory than yellow or red light. This may be one explanation for this study. A deeper understanding could be attained by also considering the relationship between rhodopsin and cryptochrome 2.

Line 79-81

This could explain why blue light irradiation exerts a stronger effect on learning and memory ability than white light, yellow light, or red light, as mice are most sensitive to the range of UV to blue light to green light (21).

Reference 21

21. Jacobs, G.H.; Neitz, J.; Deegan, J.F. Retinal eceptors in rodents maximally sensitive to ultraviolet light. Nature 1991, 353, 655–656.

 

4. Line 72: Does the light emitted by the fluorescent lamps contain blue light? If yes, how would we overcome this confounding factor?

Response: Thank you for your comment. Blue light is included in all light. However, the energy content of blue light in fluorescent lamps is actually less than 1 kJ/m², which is considered negligible.

 

5. Line 193-194: Were other factors that impact cytokine levels in the brain, or conditions that cause neuro-inflammation, taken into account when measuring the cytokine levels?

Response: Thank you for your comment. To measure brain cytokine levels, our laboratory collects the brain quickly, homogenizes it into a tissue suspension, and then immediately measures relevant levels.

 

6. Line 212: Ag-1 should be corrected to Arg-1

Response: Thank you for highlighting this error. We corrected Ag-1 to Arg-1.

Line 225

Arg-1

 

7. Line 232: Could you please clarify the distinction between M1 and M2 macrophages in your study? It's generally understood that M2 macrophages have anti-inflammatory properties, while inflammatory cytokines such as TNF-alpha and IL-6 are usually secreted by M1 macrophages. Clarifying this distinction would help in better understanding the role of macrophages in your findings.

Response: Thank you for your comment. We have added a brief description of M1 and M2 macrophages.

Line 248-250

Macrophages are typically divided into type 1 and type 2. M1 macrophages secrete inflammatory cytokines such as TNF-α and IL-6, whereas M2 macrophages secrete anti-inflammatory cytokines such as IL-4 and TGF-β (41).

Reference 41

41. Mills, C.D.; Kincaid, K.; Alt, J.M.; Heilman, M.J.; Hill, A.M. M-1/M-2 macrophages and the Th1/Th2 paradigm. Immunol. 2000, 164, 6166–6173.

 

8. Line 219: ?Mnal1 -> Bmal1

Response: Thank you for your comment. Sorry, it was our mistake. We corrected Mnal1 to Bmal1.

Line 232

Bmal1

 

9. Line 237: “Microglia shift to M1 macrophages and directly or indirectly induce the secretion of inflammatory cytokines…:

Response: Thank you for your comment. We removed “and”.

Line 274

and - delete

Reviewer 2 Report

Comments and Suggestions for Authors

I appreciate all the hard work the study group invested in this research, which was based on their previous experience in the field. Their findings are important and based on reproducible methods. The manuscript appears well organized, and generally with attention to details. I have some comments/questions/suggestions:

1.       Abstract:

a.       Please state clear the AIM (OBJECTIVE) of your study, not what you investigated.

b.       Please mention how many mice, age and sex, as well as number of groups, including control group.

c.        Please insert how did you asses the “decline in memory and learning ability”, as it was written in the main manuscript. The Abstract should summarize the whole paper.

 2.       Keywords: It would be advisable to use other Keywords, not just those belonging to the title. This would increase the likelihood of the paper being found by readers. The importance of Keywords is to improve indexing.

 3.       Introduction:

a.       Reference [12] does not have anything to do with diabetes. It is just a study protocol, published in 2015. Please update, with results and also insert a correct reference. There are plenty recent ones.

b.       Reference [13] represents a review, not a proper study referring to arterial hypertension. Please here and everywhere in the whole text, revise all references and include the correct and original ones. There is a plethora of recent ones.

c.        Line 49 – please add, after learning abilities”: “in mice”.  

d.       Same for line 52 - reference [20], by the authors: “in mice”

e.       “We also investigated the biochemical and immunological changes in the brain caused by long-term blue light irradiation”. This would be a secondary aim.

 4.       Materials and Methods

a.       Line 60 – age appears to be 6 weeks, while in Figure 1 it is written 10 weeks of age. Please clarify.

b.       Lines 86-87 – please correct to “device”.

c.        Line 89 – please explain why just “one mouse”.

 5.       Discussion:

a.       This paragraph is too short, given the results. It should be expanded more, with comments regarding the own findings and the recent literature.

b.       Limitations should be included here, not in Conclusion.

c.        PROPER directions for future research should also be inserted here, not in Conclusion.

d.       Please include in what way your findings are important, including novelties, as you did not emphasize properly your work.

 6.       Conclusion should be rewritten. Figure 6 should be inserted in Discussion, not in Conclusion.

Comments on the Quality of English Language

Generally good; minor editing of the English language is required.

Author Response

Response to the Reviewers’ comments

 

To Reviewer 2

Thank you very much for your detailed and valuable comments. We have carefully considered the reviewer's point of view and addressed each as follows.

1.Abstract:                                                                                                       a.Response: Thank you for your comment. We clearly stated the purpose of the study.

Line 13-15

Herein, we examined the effects of widespread blue light exposure on the learning and memory abilities of blue light-exposed mice.

b. Response: Thank you for your comment. We added the number of mice used, their age, sex, and number of groups.

Line 15-16

Ten-week-old male ICR mice were divided into 5 groups (5 mice/group) and irradiated with blue light from a light-emitting diode daily for 6 months.

c.Response: Thank you for your comment. We measured memory learning ability using the Morris water maze test and the step-through passive avoidance test.

Line 17-18

After 6 months of blue light irradiation, mice exhibited a decline in memory and learning abilities, assessed using the Morris water maze and step-through passive avoidance paradigms.

 

2. Keywords:

Response: Thank you for your comment. The following keywords have been added.

Line 26-27

blue light; brain and muscle arnt-like 1; inducible nitric oxide synthase; arginase-1; angiopoietin-like protein 2; interleukin-6

 

3. Introduction:

a.Response: Thank you for your comment. We replaced reference 12.

Reference 12

12. Dumpala, S.; Zele, A.J.; Feigl, B. Outer retinal structure and function deficits contribute to circadian disruption in patients with type 2 diabetes. Invest. Ophthalmol. Vis. Sci. 2019, 60, 1870–1878.

b.Response: Thank you for your advice. Reference 13 has been replaced with the original paper.

Reference 13

13. Bryk, A.A.; Blagonravov, M.L.; Goryachev, V.A.; Chibisov, S.M.; Azova, M.M.; Syatkin, S.P. Daytime exposure to blue light alters cardiovascular circadian rhythms, electrolyte excretion and melatonin production. Pathophysiology 2022, 29, 118–133.

c.Response: Thank you for your advice. We have added “mice” as suggested, editing the statement as follows.

Line 50

in memory and learning abilities in mice (19).

d.Response: Thank you for your advice. We have added “mice” as suggested, editing the statement as follows.

Line 53-54

Moreover, mice subjected to long-term UVA ocular irradiation exhibited hippocampal degeneration (20).

e.Response: Thank you for your advice. We have edited and rewritten the "We also......light irradiation" sentence.

Line 57-59

Accordingly, the purpose of this study was to determine the effects of daily blue light exposure over a 6-month period on the memory and learning abilities of mice and elucidate how blue light affects the brain.

 

4. Materials and Methods:

a.Response: Thank you for your advice. We apologize for the error; we used 10-weel-old mice.

Line 62

10-week-old male

b.Response: Thank you for your advice. We apologize for the error; the term has been altered to “device”.

Line 92

device

c.Response: Thank you for your comment. The locomotor activity measurement device uses a sensor to measure the number of times the mouse moves a certain distance. Therefore, only one animal can be measured at a time.

Line 97-99

In the current study, the locomotor activity measuring device used a sensor to measure the number of times the mouse moved a certain distance, allowing only individual measurements at a time.

 

5. Discussion

a.Response: Thank you for your advice. We have enriched the Discussion part as follows:

Line 216-219

Consistently, we found that blue light irradiation could induce a decrease in Bmal1 expression and an increase in Cry1/2 expression (Figure 3). Furthermore, an increase in M1 type macrophage was also observed (Figure 4). Although this study did not directly investigate the signal transduction between Bmal1 and M1 type macrophages,

Line 235-247

On the other hand, Angptl2 is induced during tissue damage repair and is crucial for maintaining homeostasis in the body (34,35). Furthermore, Angptl2 promotes the degradation of inhibitor of nuclear factor-κB (I-κB), a nuclear factor-κB (NF-κB ) suppressor gene, through α5β1 integrin, causing nuclear translocation of NF-κB and activating the expression of inflammation-related genes (36,37). As a result, Angptl2 promotes the activation of p38 mitogen-activated kinase (MAPK) and provokes the expression of matrix metalloproteinases (MMPs) (36,38). In the current study, blue light irradiation increased Angptl2 levels in the hippocampus (Figure 5). Furthermore, abundant Angptl2 expression has been detected in macrophages (39). High expression levels of Angptl2 in macrophages accumulated within lesions induce a hyperreactive state of tissue repair mechanisms, such as activation of MMPs and chronic inflammation (40). In the current study, the increased hippocampal levels of Angptl2 suggest that Angptl2 enhanced inflammatory cytokines in hippocampal glial cells.

Reference 33-37

34. Motokawa, I., Endo, M.; Terada, K.; Horiguchi, H.; Miyata, K.; Kadomatsu, T.; Morinaga, J.; Sugizaki, T.; Ito, T.; Araki, K.; Morioka, M.S.; Manabe, I.; Samukawa, T.; Watanabe, M.; Inoue, H.; Oike, Y. Interstitial pneumonia induced by bleomycin treatment is exacerbated in Angptl2-deficient mice. Am. J. Physiol. Lung Cell Mol. Physiol. 2016, 311, L704–L713.
35. Horiguchi, H.; Motoyoshi, E.; Kawane, K.; Kadomatsu, T.; Terada, K.; Morinaga, J.; Araki, K.; Miyata, K.; Oike, Y. ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis. EMBO J. 2017, 36, 409–424.
36. Kadomatsu, T.; Endo, M.; Miyata, K.; Oike, Y. Diverse roles of ANGPTL2 in physiology and pathophysiology. Trends Endocrinol. Metab. 2014, 25, 245–254.
37. Tabata, M.; Kadomatsu, T.; Fukuhara, S.; Miyata, K.; Ito, Y.; Endo, M.; Urano, T.; Zhu, H.J.; Tsukano, H.; Tazume, H.; Kaikita, K.; Miyashita, K.; Iwawaki, T.; Shimabukuro, M.; Sakaguchi, K.; Ito, T.; Nakagata, N.; Yamada, T.; Katagiri, H.; Kasuga, M.; Ando, Y.; Ogawa, H.; Mochizuki, N.; Itoh, H.; Suda, T.; Oike, Y. Angiopoietin-like protein 2 promotes chronic adipose tissue inflammation and obesity-related systemic insulin resistance. Cell Metab. 2009, 10, 178–188.
38. Odagiri, H.; Kadomatsu, T.; Endo, M.; Masuda, T.; Morioka, M.S.; Fukuhara, S.; Miyamoto, T.; Kobayashi, E.; Miyata, K.; Aoi, J.; Horiguchi, H.; Nishimura, N.; Terada, K.; Yakushiji, T.; Manabe, I.; Mochizuki, N.; Mizuta, H.; Oike, Y. The secreted protein ANGPTL2 promotes metastasis of osteosarcoma cells through integrin a5b1, p38 MAPK, and matrix metalloproteinases. Sci. Signal 2014, 7, ra7.

b.Response: Thank you for your advice. Limitations has been moved to the Discussion section.

c.Response: Thank you for your advice. Proper directions for future research has been moved to the Discussion section.

d.Response: Thank you for your advice. The novelty and importance of the study have been stated in the Discussion section.

Line 274-282

Collectively, the results of the present study revealed that blue light regulates clock genes in the brain and. induces microglial activity. Microglia shift to M1 macrophages, directly or indirectly induce the secretion of inflammatory cytokines, leading to a decline in learning and memory (Figure 6). These results indicate that staring directly at blue light from smartphones and computers for prolonged periods of time can damage the brain and suggest the need to protect the eyes from blue light. However, we only conducted experiments in mice. Mice are nocturnal animals, and the expression of clock genes is not identical to that in humans. Therefore, it is necessary to conduct clinical trials and further assessments using human subjects.

 

6.Conclusion

a.Response: Thank you for your advice. We removed the conclusion part. Figure 6 has been moved to the Discussion section.

Reviewer 3 Report

Comments and Suggestions for Authors

Overall, the presented experiment is interesting and some of the data appear very striking. The question is relevant and prior data studying effects of blue light on brain physiology make an influence of blue light on mouse behavior plausible. I have the following concerns:  

 

1. It appears the red, green and blue light had the same energy (40kJ/m2), but the white light used as control from Figure 2 onwards had substantially lower energy (12kJ/m2). How does this affect temperature in the cages? Do mice have a sympathetic reaction to heat stress in the blue light group but not in the white light group and control group? If material from the red and green group has been stored, repeating some of the assays in Figure 3-5 with this group could alleviate this concern, as the energy was the same as blue light. 

 

2. How often was this experiment repeated? It sounds as if one experiment is depicted. It seems this is quite a complicated experiment to perform, but having at least one repeat experiment would strengthen the findings. 

 

3. Why do mice have to be housed alone for this experiment? It appears blue light has been studied in the context of depression and has been proposed to alleviate or aggravate symptoms of depression. Mice are social animals that can have depression symptoms when housed alone. Could blue light elicit its effect on experiments in Figure 2 by aggravating depressive symptoms? This should be discussed and the rationale for housing animals alone should be explained if I didn't miss it.

 

4. Expression of genes cannot be taken for an increase in cell numbers. Higher Arg-1 levels could be due to higher numbers of Arg-1 expressing cells or upregulation of Arg-1 in the same number of cells. It would be preferable to perform additional flow cytometric analysis from hippocampal tissues to study the mechanism of higher inflammatory gene expression. Since that is likely not possible, the sentences which suggest gene expression levels demonstrate a change in cell numbers should be revisited and alleviated. (e.g. lines 203/204)

 

5. Discussion should include more hypotheses how blue light leads to the putative changes in inflammatory gene expression. Does melatonin affect immune cells in this context and how? Does blue light affect mood and stress levels of the animals and glucocorticoid/adrenergic signaling in immune cells affect putative immune cell composition and performance in behavioral tests?

Minor points:

In methods 2.6 the title says “Western blotting analysis of the ciliary muscle”, is this a mistake? It seems Hippocampus samples were analyzed. 

“Expression of microglia” line 179

“Agr-1” in line 183

Line 192 hip pocampus

Line 212: “Ag-1”

There are multiple full stops (.) in the text, for example in line 232.

 

 

Author Response

Response to the Reviewers’ comments

 

To Reviewer 3

Thank you very much for your detailed and valuable comments. We have carefully considered the reviewer's point of view, with each addressed as follows.

 

1. Response: Thank you for your crucial comment. In this test, the room temperature was kept constant and air was circulated in the irradiation cage. Therefore, there was no change in temperature. As stated, analyzing the substances in the red light and green light groups would be valuable; however, the data were not stored and cannot be analyzed. Henceforth, we plan to preserve and analyze substances of each color.

 

2. Response: Thank you for your valuable opinion. The test was performed once. As stated by the reviewer, considerable effort is required to run this test. There are also individual differences between mice. Therefore, multiple further iterations could yield more obvious results. In future experiments, we will try to increase the number of repetitions when conducting the test.

 

3. Response: Thank you for your important points. In this experiment, the mice were housed in each group and not individually. This description will be clarified in the manuscript. Thank you for pointing it out.

 Line 63-65

Mice groups were housed in cages in an air-conditioned room maintained at 23 ± 1°C with a 12-h light/12-h dark cycle under SPF and stress-free conditions.

 

4. Response: Thank you for your important comments. As stated, we did not measure the number of cells properly. Therefore, in all cases, we will change the expression to ``there was a tendency to decrease or increase.''

 

5. Response: Thank you for your important comments. We will add reported hypotheses about how blue light can alter inflammatory gene expression to the Discussion section. Regarding melatonin, melatonin is related to circadian rhythms and may affect immune cells. However, there have been reports that mice, which are experimental animals, are unable to synthesize melatonin, and we are currently investigating whether the melatonin synthesis occurs (Goto M., et al. J. Pineal Res. 1987, 7, 195-204.; Kasahara T., et al. Proc. Natl. Acad. Sci. USA. 2010, 107, 6312-6417.).

Line 258-273

Blue light has also been found to induce stress in the brain. Living organisms feel stress when exposed to blue light. Stress causes the secretion of stress hormones, glucocorticoids. Glucocorticoids reportedly inhibit new synapse formation and result in memory inhibition. Furthermore, in the relationship between brain microglia and stress, stress causes a rapid increase in brain epinephrine levels and enhances glucocorticoid levels in the brain (46,47). In the brain, epinephrine acts through α- and β-adrenergic receptors to regulate learning and memory. Glucocorticoids easily enter the brain and regulate learning and memory via glucocorticoid receptors (48,49). In the brain, microglia are crucial regulators of neurological function (50,51) and comprise several receptors for epinephrine and glucocorticoids (52). Stress increases the number of microglia, impairing crosstalk with neurons and disrupting glutamate signaling (53). Considering the immune response of microglia, stress induces a pro-inflammatory state in microglia, enhancing the expression of IL-1, IL-6, and TNF-α (54) and reducing levels of the anti-inflammatory cytokine IL-10 (55). Accordingly, stress may cause memory impairment and related symptoms. Given that stress was not analyzed in the current study, it is necessary to also consider the blue light/stress axis.

References 46-55

46. Sara, S.J. The locus coeruleus and noradrenergic modulation of cognition. Nat. Rev. Neurosci. 2009, 10, 211–223.
47. De Kloet, E. R.; Joels, M.; Holsboer, F. Stress and the brain: From adaptation to disease. Rev. Neurosci. 2005, 6, 463–475.
48. Bahtiyar, S.; Karaca, K.G.; Henckens, M.J.A.G.; Roozendaal,　 Norepinephrine and glucocorticoid effects on the brain mechanisms underlying memory accuracy and generalization. Mol. Cell Neurosci. 2020, 108, 103537.
49. Joels, M.; Sarabdjitsingh, R.A.; Karst, H. Unraveling the time domains of corticosteroid hormone influences on brain activity: Rapid, slow, and chronic modes. Rev. 2012, 64, 901–938.
50. Wu, Y.; Dissing-Olesen, L.; MacVicar, B.A.; Stevens, B. Microglia: Dynamic mediators of synapse development and plasticity. Trends Immunol. 2015, 36, 605–613.
51. Morris, G. P.; Clark, I.A.; Zinn, R.; Vissel, B. Microglia: A new frontier for synaptic plasticity, learning and memory, and neurodegenerative disease research. Learn. Mem. 2013, 105, 40–53.
52. Sierra, A.; Gottfried-Blackmore, A.; Milner, T.A.; McEwen, B.S.; Bulloch, K. Steroid hormone receptor expression and function in microglia. Glia 2008, 56, 659–674.
53. Rogers, J. T.; Morganti, J.M.; Bachstetter, A.D.; Hudson, C.E.; Peters, M.M.; Grimming, B.A.; Weeber, E.J.; Bickford, P.C.; Gemma, C. CX3CR1 deficiency leads to impairment of hippocampal cognitive function and synaptic plasticity. Neurosci. 2011, 31, 16241–16250.
54. Han, Y.; Zhang, L.; Wang, Q.; Zhang, D.; Zhao, Q.; Zhang, J.; Xie, L.; Liu, G.; You, Z. Minocycline inhibits microglial activation and alleviates depressive-like behaviors in male adolescent mice subjected to maternal separation. Psychoneuroendocrinology 2019, 107, 37–45.
55. Voorhees, J. L.; Tarr, A.J.; Wohleb, E.S.; Godbout, J.P.; Mo, X.; Sheridan, J.F.; Eubank, T.D.; Marsh, C.B. Prolonged restraint stress increases IL-6, reduces IL-10, and causes persistent depressive-like behavior that is reversed by recombinant IL-10. PLoS One 2013, 8, e58488.

 

Minor points

1. Thank you for your advice. We apologize for the error, we meant to imply “hippocampus”.

Line 134

hippocampus

2. Thank you for your advice. Changed to “Expression of microglia”.

Line 187

expression of microglia

3. Thank you for your advice. We apologize for the error, we meant to imply the “Arg-1”.

Line 191

Arg-1

4. Thank you for your advice. We apologize for the error, we meant to imply “hippocampus”.

Line 200

hippocampus

5. Thank you for your advice. We apologize for the error, we meant to imply the “Arg-1”.

Line 225

Arg-1

6. Thank you for your advice. Removed extra full periods (.) in the text.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for your responses. While some of my concerns have been sufficiently addressed, others are in my opinion not yet resolved: 

 

1. If it was not necessary to house mice individually, repeating the experiment appears feasible under normal circumstances. Having no repeat experiment is a severe limitation of a study. I think that at the very least it has to be very clear that the presented data are representative only from one experiment. This should be mentioned both in the methods and the discussion segment, where other limitations of the study are discussed (e.g. mice as nocturnal animals.)

2. I am not content with the rephrasing of the section about immune cell related gene expression in the brain tissue. If I understand correctly, the expression of genes measured via qPCR is taken as a surrogate of cell numbers in the tissue. As I have written in the first review, this is not common and the text must be rephrased. For example (example suggestion in bold)

Next, we examined  expression of genes correlated with immune cell populations  that are involved in memory and learning.

(...)

Furthermore, macrophages are di- vided into two types: M1 and M2. Blue light irradiation increased levels of CCR7 and iNOS, which are representative expressed by of M1 macrophages (Figure 4B, 4C, 4D). In contrast, the level of Arg-1, a representative gene expressed by of M2 macrophages, was decreased by blue light irradiation (Figure 4E). This data could indicate an altered M1/M2 ratio in the hippocampus after blue light exposure. 

 

Moreover, I do not understand this sentence and the corresponding data:

 

Blue light irradiation for 6 months increased the expression of microglia, 188 which are hippocampal macrophages (Figure 4A, 4C). 

194 

 

It appears the data show Iba-1 expression, a gene that is expressed by microglia. The graph 5A must state "Iba-1/b-Actin" and cannot be titled "microglia/b-Actin". Again, the authors can suggest that this could mean that there are more microglia, but gene expression data alone cannot prove that it is not the same amount of microglia who express higher levels of Iba-1 (or an entirely different cell type expressing Iba-1). 

 

In the discussion, the difference between marker gene expression and cell abundance also needs to be clear. (e.g. "Furthermore, an increase in M1 type macrophage was also observed")

 

Minor: there is an additional "." in line 276

Comments on the Quality of English Language

The phrasing of gene expression versus cell abundance needs to be made more clear. 

Author Response

Response to the Reviewers’ comments

 

To Reviewer 3

Thank you very much for your detailed and valuable comments. We have carefully considered the reviewer's point of view, with each addressed as follows.

 

1. Response: There is no excuse. It's a spelling mistake in English. This study was retested once and the same results were obtained. However, the test period for this study is long, 6 months, and it is difficult to retest. We are currently starting further trials to search for reagents with improved effects.

Line 83-84

In addition, this study was retested once and the results were equivalent.

 

2. Response: Thank you for your valuable opinion. As shown by the reviewer, the measured gene expression is interpreted as a surrogate for the number of cells in the tissue. Therefore, I have rewritten it as suggested by the reviewer.

Line 186-195

3.3. Effect of long-term blue light irradiation on Iba1, CCR7, iNOS, and Arg-1 levels in the hippocampus

Next, we examined expression of genes correlated with immune cell populations that are involved in memory and learning. Blue light irradiation for 6 months increased the expression of Iba1, which are hippocampal macrophages (Figure 4A, 4C). Furthermore, macrophages are divided into two types: M1 and M2. Blue light irradiation increased levels of CCR7 and iNOS, which are expressed by M1 macrophages (Figure 4B, 4C, 4D). In contrast, the level of Arg-1, a gene expressed by M2 macrophages, was decreased by blue light irradiation (Figure 4E). This data could indicate an altered M1/M2 ratio in the hippocampus after blue light exposure.

Response: As the reviewer showed, gene expression data alone cannot prove that it is not the same amount of microglia who express higher levels of Iba-1 (or an entirely different cell type expressing Iba-1). Therefore, we changed the microglia to Iba-1.

Response: Thank you for your important points. Our study showed an increase in M1 type macrophages.

Line 257

Furthermore, an increase in M1 type macrophage was also observed (Figure 4).

 

Minor comment:

Removed extra full periods (.) in the text.

Line 278