Abstract

   Myopia is a substantial global public health concern primarily linked
   to the elongation of the axial length of the eyeball. While numerous
   animal models have been employed to investigate myopia, the specific
   contributions of genetic factors and the intricate signaling pathways
   involved remain incompletely understood. In this study, we conducted
   RNA-seq analysis to explore genes and pathways in two distinct
   myopia-inducing mouse models: form-deprivation myopia (FDM) and
   lens-induced myopia (LIM). Comparative analysis with a control group
   revealed significant differential expression of 2362 genes in FDM and
   503 genes in LIM. Gene Set Enrichment Analysis (GSEA) identified a
   common immune-associated pathway between LIM and FDM, with LIM
   exhibiting more extensive interactions. Notably, downregulation was
   observed in OxPhos complex III of FDM and complex IV of LIM. Subunit A
   of complex I was downregulated in LIM but upregulated in FDM.
   Additionally, complex V was upregulated in LIM but downregulated in
   FDM. These findings suggest a connection between alterations in energy
   metabolism and immune cell activation, shedding light on a novel avenue
   for understanding myopia’s pathophysiology. Our research underscores
   the necessity for a comprehensive approach to comprehending myopia
   development, which integrates insights from energy metabolism,
   oxidative stress, and immune response pathways.

   Keywords: myopia, RNA sequencing, animal model, mice

1. Introduction

   Myopia, or near-sightedness, is the world’s most common eye disorder
   and is caused by light focusing in front of the retina, typically
   caused by axial elongation of the eye during development and into early
   adulthood [[44]1]. While it is relatively easy to treat with corrective
   lenses to improve vision, myopia itself is a factor that increases the
   risk of developing eye diseases such as glaucoma, retinal detachment,
   and cataracts [[45]2,[46]3].

   Mechanisms of myopia development have been investigated in animal
   models; however, the exact mechanism is still unclear
   [[47]4,[48]5,[49]6]. Representative methods for inducing myopia in
   animal models include the form-deprivation myopia (FDM) model, which
   employs a translucent diffuser, and the lens-induced myopia (LIM)
   model, achieved by imposing defocus through a negative lens causing
   hyperopic defocus, both of which commonly result in axial elongation
   [[50]7]. The mechanism underlying this structural change is theorized
   to be emmetropization, a developmental process that ensures the eye’s
   optical power matches its axial length, allowing clear focus on the
   retina.

   Previous studies have identified possible differentially expressed
   genes and pathways involved in myopia models
   [[51]8,[52]9,[53]10,[54]11]. However, there is not much of a consensus
   among the existing studies due to variations in animal models and
   experimental designs. Additionally, they have uncovered biochemical
   signaling pathways that translate defocus-related visual stimuli into
   retinal cellular and biochemical changes, which then signal changes in
   the retinal pigment epithelium (RPE), choroid, and ultimately sclera,
   resulting in altered eye growth and refractive state changes. However,
   the differences between FDM and LIM models have not been clearly
   reported.

   There may be a difference between the FDM model and the LIM model in
   the mechanism that causes myopia. Research conducted on chicks has
   revealed that the dopamine mechanisms responsible for mediating the
   protective effects of brief periods of unrestricted vision might vary
   between the FDM and LIM models [[55]12]. This suggests a potential
   divergence in the mechanisms governing growth control between these two
   methods. However, to our best knowledge, there are limited reports
   about the differences between these two forms of myopia models with the
   same experimental conditions, such as experimental duration and
   environment.

   In the present study, we compare FDM and LIM in mice under the same
   experimental conditions to identify common genes and pathways involved
   in the development of myopia differential expression analysis and
   pathway analysis with mice RNA data from these two myopia models and a
   control.

2. Materials and Methods

2.1. Animals

   All procedures adhered to the Association for Research in Vision and
   Ophthalmology (ARVO) statement on the use of animals in ophthalmic and
   vision research and were approved by the animal institutional review
   board of Kyung Hee University hospital at Gangdong (KHNMC AP 2022-014).
   Nine male three-week-old Balb/c mice weighing 10–15 g were used in this
   study. Three mice were assigned to each experimental group (three for
   the control, three for LIM, three for FDM). After being anesthetized
   via an intramuscular injection of alpaxalone (10 mg/mL, Alfaxan, Jurox,
   Kansas City, MO, USA), in the LIM group, a −10.0 diopter (D) lens was
   affixed to a frame created through the processing of an Eppendorf tube.
   This frame was subsequently secured onto the left eye utilizing 8-0
   prolene sutures on animal skin ([56]Figure 1). In the FDM group, the
   left eye was sutured using 8-0 prolene. The control group did not
   receive any form of treatment. After four weeks, the animals were
   euthanized using a CO[2] chamber and enucleated. Total RNA was
   extracted from their whole eyes.

Figure 1.

   [57]Figure 1
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   Form-deprivation myopia (A) and lens-induced myopia (B) in mice.

2.2. RNA-Seq Preprocessing

   Raw datasets (i.e., fastq) of nine samples in total, where three were
   from the LIM model, three from the FDM model, and the other three from
   the control group, were acquired from bulk RNA sequencing. Through the
   sequencing process, paired-end reads whose length was 101 bp were
   generated for all the samples using the illumina platform with the
   truseq stranded mRNA library prep kit. Next, we applied a preprocessing
   procedure; (i) alignment: STAR aligner (v2.7.3a) was executed [[59]13];
   (ii) quantification: HTSeq-count (v0.12.4) was implemented [[60]14];
   (iii) normalization: VST (Variance Stabilizing Transformation) was
   applied using the DESeq2 package [[61]15]. The reference genome,
   GRCm39, with its annotation was utilized in the mapping for all the
   reads of the three conditions.

2.3. Differential and Enrichment Analysis

   We performed two different gene expression profile comparisons such as
   ‘LIM vs. control’ and ‘FDM vs. control’ using the DESeq2 package (p <
   0.05) whose visualization was implemented using EnhancedVolcano
   [[62]16] and clusterProfiler [[63]17]. Four different gene sets were
   used for the enrichment analysis with gene ontology terms; (i) all
   aligned genes, (ii) genes only associated with eyes, (iii) genes only
   associated with the immune system, and (iv) genes only associated with
   inflammation. These gene groups were selectively listed using the
   Molecular signature database (MSigDB) with corresponding keywords
   [[64]18,[65]19,[66]20]. Pathway enrichment analysis through Gene Set
   Enrichment Analysis (GSEA) was executed and displayed using Cytoscape
   [[67]21] along with its visualization app EnrichmentMap [[68]22]. From
   the DEGs, Protein–Protein Interaction (PPI) networks were produced,
   where each interaction score was higher than 0.9, through STRING
   [[69]23], which is a protein interaction database including known
   direct or indirect associations. Another pathway enrichment analysis
   was conducted through Fast Gene Set Enrichment Analysis (fgsea)
   [[70]24] utilizing DEGs of MitoCarta 3.0 [[71]25]. In these analyses,
   five genes (Alas2, Hbb-bs, Hbb-bt, Hba-a1, Hba-a2) which are marker
   genes of red blood cells were removed from the downstream analysis to
   consider possible contamination with blood.

2.4. Analysis on Mitochondria-Associated Expression Profiles

   The custom-made gene list of mitochondrial regulatory mechanisms in
   [[72]26] was used to selectively explore gene expression profiles
   associated with mitochondrial energy metabolism across
   mitochondria-associated pathways. Gene lists of mitochondrial complexes
   and additional lists of energy metabolism-associated pathways were
   applied. To visualize gene regulations involved in the KEGG pathway of
   “oxidative phosphorylation”, the Pathview package of R was used
   [[73]27].

3. Results

3.1. Eye-Associated Differentially Expressed Genes between LIM and FDM Are
Different

   To explore the functional association of transcriptional profiles of
   LIM and FDM, we first compared them with the control and identified 503
   genes from LIM and 2362 genes from FDM that were significantly
   differentially expressed. As shown in [74]Figure 2A, some overlapping
   differentially expressed genes (DEGs) including Ctag2l2, Gm8618,
   S100a9, and Cxcl5 exhibited upregulation in both LIM and FDM, while
   Krtap8-1, Oas1f, Krt71, Krt331, Krt72, Dmbt1, Them5, Bpifc, and Abca12
   were commonly downregulated in both of them. Among those genes,
   Ctag2l2, whose human ortholog, CTA1A, was one of the human myopia
   candidate genes [[75]28] and S100a9, known to be implicated in the
   regulation of the immune response, was previously identified to be
   upregulated in a guinea pig FDM model [[76]10]. We further inquired
   into eye-specific functional associations by performing Gene ontology
   (GO) analysis using only eye-associated genes obtained from MSigDB
   ([77]Figure 2B). While signaling pathways such as response to stimulus,
   signal transduction, structure formation in morphogenesis, and
   epithelial cell differentiation were listed as the top four
   significantly changed pathways in LIM, FDM exhibited significant
   alterations on pathways such as neuron projection, plasma membrane
   bounded cell projection organization, neuron development, and nervous
   system process at the top.

Figure 2.

   [78]Figure 2
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   Differentially expressed genes and enriched pathways. (A)
   Differentially expressed genes in LIM and FDM groups compared to the
   control group (p-value < 0.05 and log2 fold-change > 2). (B) Top 20
   most enriched pathways of known eye-associated genes from MSigDB in LIM
   and FDM compared with the control (p-value < 0.1 and q-value < 0.25).
   (C) Top 10 most enriched pathways directly associated with immune and
   inflammation in LIM and FDM compared with the control (p-value < 0.1
   and q-value < 0.25).

   Other pathways, however, were found to be directly or indirectly
   associated with immunologic pathways in both LIM and FDM, while
   upregulation of immune-associated GO terms has been observed in
   previous studies [[80]29,[81]30] and the importance of inflammation in
   myopia progression has been proven clinically and experimentally
   [[82]31]. To further look into this association, we performed GO
   analysis using only either immune-associated or inflammation-associated
   genes selected from MSigDB. T cell activation, regulation of defense
   response, and positive regulation of immune response were revealed as
   commonly upregulated in both LIM and FDM. However, response to virus,
   defense response to symbiont, viral process, viral life cycle, and
   pattern recognition receptor signaling pathway were shown in common
   downregulation between LIM and FDM. For inflammation-specific pathways,
   while more inflammatory pathways were enriched in LIM, (positive)
   regulation of inflammatory response and acute inflammatory response
   were commonly listed up in both LIM and FDM.

3.2. GSEA & PPI-Based Clusters Exhibited Significant Alterations in
Immune-Associated and Energy Metabolism

   To determine the underlying biological mechanisms of myopia, we
   implemented Gene Set Enrichment Analysis (GSEA) to further explore
   interactions between pathways as illustrated in [83]Figure 3A,B.
   Primarily overlapping pathway clusters between LIM and FDM turned out
   to be immune-associated clusters, where LIM exhibited more extensive
   interactions encompassing not only lymphocyte regulations, such as
   regulation of B or T cells, whereas myeloid cell regulations such as
   granulocyte migration, granulocyte chemotaxis, and neutrophil
   migration. The humoral immune response featured multiple interactions
   with B and T cell responses, suggesting that its complement system
   might be involved in modulating the immune response in myopia, as
   previous studies presented that patients with myopia have an elevation
   of complement components in their serum and aqueous humor
   [[84]32,[85]33,[86]34]. LIM also disclosed LIM-specific
   immune-associated clusters including T cell selection, immune response
   regulating signaling pathway, immune receptor activity, phagocytosis,
   and myeloid cell differentiation. Only one cluster, skin development,
   comprising intermediate filament, keratin filament, keratinocyte
   differentiation, and keratinization was in downregulation in LIM.
   Several clusters that could be possibly associated with eye development
   such as photoreceptor cell development, sensory perception of light
   stimulus, and mitochondrial localization were suppressed in FDM.
   Specifically, the suppression of mitochondrial energy metabolism along
   with the activation complement system aligns with the findings of
   previous studies on myopia development [[87]35,[88]36,[89]37].

Figure 3.

   [90]Figure 3
   [91]Open in a new tab

   GSEA-based network visualization in LIM and FDM groups compared to the
   control group. (A) Network association of LIM compared with the
   control. (B) Network association of FDM compared with the control.
   Edges are inferred by considering strong correlation between signaling
   pathways and each node (as enriched pathway) size corresponds with
   number of edges. The color is proportional to degree of
   differentiation, where up/downregulation indicates red or blue,
   respectively. The selected pathways were filtered using false discovery
   rate (FDR) < 0.75 and edge similarities > 0.375.

   To explore other potential functional associations, we searched for any
   potential clusters on the Protein–Protein Interaction (PPI) network
   between LIM and FDM using STRING and visualized them using Cytoscape.
   The primary revelation from this PPI-based analysis was metabolic
   processes in downregulation such as the small molecule metabolic
   process or the nucleic acid metabolic process shown in [92]Figure 4 as
   commonly downregulated. This observation is in line with the
   aforementioned finding in downregulation through GSEA since metabolic
   processes are associated with mitochondrial energy metabolism. Other
   commonly up/downregulated clusters turned out to be at partially at
   least associated with pathways of immune processes as already
   demonstrated in significant alterations analyzed through GSEA.
   Additionally, myopia-model-specific up/downregulation included protein
   interactions of protein/cell adhesion such as hemidesmosome assembly or
   cell–cell adhesion in downregulation, and visual perception in
   upregulation for LIM. Also, protein interactions of visual perception
   in downregulation and membrane-associated transport or cellular
   processes such as endocytosis or desmosome in upregulation for FDM were
   represented. The different alteration pattern of visual perception
   could be interpreted as a compensatory mechanism using different sets
   of genes. Considering the downregulations of mitochondrial energy
   metabolism and some metabolic processes through GSEA and PPI-based
   analyses might suggest the probable direct/indirect association between
   a broad spectrum of alterations in energy metabolism and myopia
   development.

Figure 4.

   [93]Figure 4
   [94]Open in a new tab

   Protein–Protein Interaction (PPI) networks based on differentially
   expressed genes (DEGs) in LIM and FDM groups compared to the control
   group. Each node represents a protein mapped to one of the DEGs, and
   the edges represent strong interactions between corresponding proteins.
   DEGs were selected through p-values of 0.2 for LIM and 0.1 for FDM.
   Interactions with protein interaction scores higher than 0.9 were
   included into clusters where each cluster contained at least five
   proteins.

3.3. Some Energy-Associated Metabolism Pathways Demonstrated Significant
Alterations

   Since the retina is one of the most metabolically active tissues
   [[95]38], we explored transcription profiles of pathways directly or
   indirectly associated with energy metabolism to compare relative
   expression levels of specific genes of the pathways ([96]Figure 5). For
   essential pathways of energy metabolism, while fatty acid oxidation did
   not divulge considerable alterations, the TCA cycle disclosed slight
   up/down variations across its associated genes. According to studies
   from animal models or human patients [[97]39,[98]40], the TCA cycle was
   found to be one of the most impactful pathways as downregulated on
   myopia, while Cs and Ogdh could be potential target genes based on our
   analyses. Another major pathway, glycolysis, revealed significant
   alterations, but differently between LIM and FDM. This upregulation of
   glycolysis on LIM could be interpreted as upregulated glycolytic
   enzymes and pathways in LIM, as supported by a proteomics study using
   chicks [[99]41]. FDM suppression could also be considered, as a
   metabolomics study utilizing guinea pigs reported that glucose was
   found to accumulate in the form-deprived eyes, suggesting a decrease in
   aerobic glycolysis [[100]40]. In addition, previous studies discovered
   that a myopic retina experiences oxidative stress due to its hypoxic
   conditions given from the retina’s high blood flow and susceptibility
   to photic oxidative injury [[101]42]. We looked over pathways
   associated with oxidative stress such as NADPH synthesis, integrator
   stress response, and antioxidant defenses. Based on the gene expression
   patterns of those pathways, G6pd2 and Rpia in NADPH synthesis and Nme4
   and Xbp1 in integrator stress response might be potential candidate
   genes of oxidative stress on myopia.

Figure 5.

   [102]Figure 5
   [103]Open in a new tab

   Heatmap of pathways associated with mitochondrial energy metabolism.
   Transcriptional profile alterations (as t-scores) of the customized
   mitochondria-associated gene set obtained from [[104]26] were
   visualized. Red indicates upregulation in either the LIM or FDM
   compared to the control, whereas blue indicates downregulation in
   either the LIM or FDM compared to the control.

3.4. Oxidative Phosphorylation Revealed Specific Alterations

   To further investigate changes in energy metabolism, we looked into
   oxidative phosphorylation (OxPhos), the most crucial functional process
   for energy production, through three different analytic views. While
   OxPhos was generally downregulated in both LIM and FDM,
   common/different alterations across five distinct complexes were taken
   into consideration. The first analysis through fgsea with MitoCarta 3.0
   ([105]Figure 6A) displayed common suppression on OxPhos complex IV in
   both LIM and FDM. Cytochrome C was, however, upregulated in LIM while
   downregulated in FDM. In a previous study with an FDM model of guinea
   pigs, both protein and mRNA expression levels of cytochrome C protein
   (SCO1 and SCO2) were found to be considerably lower in the FDM group
   [[106]43]. The second analysis in [107]Figure 6B represented relative
   expression levels of all five complex genes. The circular heatmap
   showed common downregulation on complex I and IV in both LIM and FDM,
   but different regulation on complex V by relative downregulation in
   FDM. The third analysis ([108]Figure 6C) provided KEGG-based
   visualization of complex-associated gene expression levels. While
   downregulation occurred in complex III of FDM and complex IV of LIM,
   subunit A of complex I (NADH ubiquinone oxidoreductase) was
   downregulated in LIM while it was upregulated in FDM. Also, in complex
   V, F-type H+ transporting ATPase subunit α was upregulated in LIM while
   downregulated in FDM, and H+/K+ exchanging ATPase subunit α was
   downregulated in both LIM and FDM.

Figure 6.

   [109]Figure 6
   [110]Open in a new tab

   Multiple comparative analyses of Oxidative Phosphorylation (OxPhos) in
   LIM and FDM in comparison to the control group. (A) Up/Downregulation
   of OxPhos-associated pathways through GSEA (fgsea). Common gene lists
   between DEGs (p-value < 0.5) and MitoCarta 3.0 were used. (B)
   Transcriptional profile alteration of OxPhos with t-score. (C) OxPhos
   complex-specific gene expression changes using Pathview [[111]27]. All
   the genes used for the three different analyses were DEGs with p-value
   < 0.5.

3.5. Metabolic Alterations Occurred Extensively

   In the pursuit of prediction of metabolic alterations, we proceeded
   with a further GSEA analysis implemented through fgsea with MitoCarta
   3.0 on metabolic alterations of either LIM or FDM extensively
   ([112]Figure 7). For energy-associated metabolism other than OxPhos
   changes, pathways of LIM (NAD biosynthesis and metabolism, electron
   carriers, and carbohydrate metabolism) and coenzyme Q metabolism of FDM
   displayed significant alterations. Additionally, in FDM, mitochondrial
   permeability transition pore demonstrated sufficient change, suggesting
   disruption of mitochondrial calcium homeostasis from induction of
   myopia [[113]44]. Several other significant alterations of metabolic
   pathways that were not directly associated with energy metabolism were
   divulged, where commonly more activated metabolisms in both LIM and FDM
   turned out to be not only lipid-associated metabolism, but also
   immune-associated metabolism. For lipid metabolism, cholesterol, bile
   acid, and steroid synthesis were upregulated for both, as supported by
   previous studies which show enrichment of bile secretion and its
   associated pathways in FDM of chick models and human myopia patients
   [[114]36]. Immune-associated metabolism such as immune response,
   cAMP-PKA signaling, and itaconate metabolism disclosed relatively
   further activations in both LIM and FDM. cAMP is the core regulator of
   both pro/anti-inflammatory activities along with numerous other
   physiological processes [[115]44] and commonly upregulated pathways
   also included itaconate metabolism, which is known to play an essential
   role in immunity or inflammation [[116]45]. Additionally, LIM showed
   downregulation of chaperones in protein-related pathways which could
   imply decreased activation to deal with stress conditions during myopia
   induction. In FDM, mitochondrial central dogma with mtRNA granules and
   mt-rRNA modifications was downregulated. Modifications of mitochondrial
   rRNA ensure the stability of mitochondrial protein translation and
   dysregulation of such modifications may lead to fatal mitochondrial and
   metabolic dysfunction [[117]46]. Therefore, although different pathways
   showed alterations, disruption of mitochondrial protein homeostasis was
   present in both LIM and FDM.

Figure 7.

   [118]Figure 7
   [119]Open in a new tab

   Lollipop based on GSEA with fgsea using MitoCarta3.0 in LIM and FDM
   groups compared to the control group. (A) Enriched pathways in LIM on
   metabolism/signaling/protein import, sorting and homeostasis. (B)
   Enriched pathways in FDM on metabolism/signaling/mitochondrial central
   dogma. Common gene lists between DEGs (p-value < 0.5) and MitoCarta 3.0
   were used.

4. Discussion

   In this study on myopia, we juxtaposed both the FDM and LIM mouse
   models by employing comparable experimental settings to examine not
   only commonly associated, whereas model-specific regulatory mechanisms
   in myopia development. This investigation explored differential
   transcriptional profiles and their associated pathway activities by
   applying all gene lists, known eye-associated gene lists, and specific
   gene lists of energy metabolism-associated pathways.

   The significance of immune responses in the progression of myopia has
   been well-documented both clinically and experimentally [[120]31] and
   several studies have already noted the upregulation of
   immune-associated GO terms [[121]47]. Our GO assessment of DEGs also
   revealed an increased activation of immune-associated pathways in both
   LIM and FDM. When the analysis narrowed down to genes specifically tied
   to the immune system, both LIM and FDM frequently showed upregulation
   in pathways associated with inflammation and its positive regulation.
   Particularly, LIM displayed a more pronounced activation of pathways
   linked to immunity compared to FDM. These pathways spanned responses
   managed by T cells, as well as those linked to B cells and the humoral
   immune system. Many genes related to the immune system have been known
   to be involved in the development of myopia [[122]10], for instance,
   CTLA4 from our analysis involving T cell regulation was found to be
   uniquely upregulated in LIM, supporting a surge in the expression of
   several LIM-specific lymphocyte immune pathways. Of significant note,
   the humoral immune system saw extensive activation specifically within
   the complement system while interacting directly with B and T
   cell-driven responses, where multiple research endeavors have delved
   into the influence of the complement system on myopia [[123]48]. As
   depicted in [124]Figure 4 as protein–protein interaction-based
   clusters, the centrality of immune responses in pathway evaluations and
   their interplay was further emphasized at the proteomic level. These
   interactions were involved in systems such as chemokine receptors,
   binding of chemokine, the general immune framework, and the innate
   immunity system.

   While many previous studies have primarily focused on the retina or
   posterior parts of myopia, our study analyzed the whole eyes
   [[125]49,[126]50]. Although this approach might pose challenges for a
   detailed examination of individual ocular structures, it has enabled us
   to view a wide array of pathways associated with eye development during
   the myopia induction process simultaneously. Considering pathways
   directly/indirectly associated with visual perception, some
   discrepancies between LIM and FDM have been captured. In the FDM model,
   several pathways associated with visual perception were observed in
   downregulation and the reduction in light stimulus perception seemed
   similar to the form-deprivation technique used for inducing myopia.
   This combinatorial downregulation of the nervous system process could
   not only substantiate the modified visual perception inherent to
   myopia, it could also emphasize the significant role of contrast
   sensitivity in refractive eye development of myopia since they were
   reported to be associated with contrast sensitivity in the FDM model of
   myopia [[127]51]. LIM model clusters from the PPI network, however,
   stood in sharp contrast to GO analysis of the FDM model for the
   alterations of visual perception. This corroborates observations from
   the guinea pig LIM model, where phototransduction was highlighted as
   one of the most significant pathways [[128]52]. In addition, pathways
   associated with epithelial development exhibited contrasting behaviors:
   downregulation in LIM and upregulation in FDM. The modulation of
   epithelial development of the LIM model, particularly the regulation of
   water loss via the epithelium, suggested a potential disruption in
   water homeostasis within the eyes. This phenomenon aligns with one of
   the primary mechanisms implicated in myopia development [[129]29]. The
   observed downregulation of epithelial cells was also believed to
   reflect the alterations in soft tissues during the axial lengthening
   process, since changes in epithelium thickness in myopia are known to
   decrease in the central cornea and the central epithelium is linked
   with refractive errors [[130]53]. Therefore, the downregulation of the
   epithelial cell pathway during myopia induction likely mirrors changes
   associated with an increase in eye axial length, as well as alterations
   in refractive change.

   This study stands out by shedding light on the comprehensive changes in
   energy metabolism associated with myopia, extending beyond the insights
   provided by earlier research. One of the intriguing findings is the
   notable alterations in oxidative phosphorylation. These alterations,
   taken in conjunction with the broader shifts in energy metabolism,
   signal a potential ocular response to hypoxic damage. Hypoxia, or
   reduced oxygen supply, can prompt a cascade of metabolic and cellular
   reactions, including adaptations in energy production mechanisms.
   Several pathways closely linked to the electron transport chain were
   notably enriched in our myopia samples, indicating changes in the
   energy metabolism of eye development, as in previous research which has
   drawn attention to modifications in oxidative phosphorylation and their
   potential roles in myopia progression. A previous study using the LIM
   chick model found a significant portion of core genes in upregulated
   mitochondrial metabolism pathways to form part of complex I, III, and
   IV with additional 11 core genes from other pathways coding for complex
   V [[131]37]. Another study demonstrated that a variant of the NDUFAF7
   gene that might contribute to the development of pathological myopia by
   decreasing complex I activity resulted in decreased ATP and GTP
   production downstream, which weakened photoreceptor cell function
   [[132]54]. A meta-analysis of transcriptome data reported upregulation
   of the NDUFS5 gene in late stage of myopia, which encoded a subunit of
   complex I [[133]48]. Our results show how metabolic changes in LIM and
   FDM may differ from each other in the electron transport chain pathway.
   Oxidative phosphorylation in this study was predominantly downregulated
   in both LIM and FDM, albeit with slight variations; (i) cytochrome C:
   upregulation in LIM, but downregulation in FDM; (ii) subunit A of
   complex I: downregulation in LIM, but upregulation in FDM; (iii)
   complex IV: unique downregulation in FDM; (iv) complex III: unique
   downregulation in LIM. Other than OxPhos, the FDM model showed a
   suppression in the TCA cycle, while the LIM model exhibited broader
   metabolic changes including increased carbohydrate metabolism and
   decreased glyoxylate and glycine metabolism. Several metabolomic
   studies on animals and humans have identified the TCA cycle as a key
   pathway as downregulated in myopia [[134]39], which might be associated
   with axial length elongation [[135]55]. Furthermore, considering high
   metabolic activities of the retina, neutralization of reactive oxygen
   species that have an effect on hypoxia in myopia development might have
   a significant role in myopia development since oxidative damage to the
   retina is commonly associated with hypoxic conditions [[136]56].

   One major limitation of this study comes from its experimental design.
   The ocular growth is thought to be controlled locally by the posterior
   eye, and therefore, most myopia studies have analyzed RNA extracted
   after isolating posterior eye structures including the retina and the
   choroid. However, the RNA was extracted from the whole eye in this
   study and this may have resulted in less sensitive results.

5. Conclusions

   The association between energy metabolism alterations and immune cell
   activation might open up a new avenue for understanding the
   pathophysiology of myopia. The immune system’s role in myopia
   development has been a topic of growing interest, and our findings hint
   at a deeper interplay between metabolic changes and immune responses.
   It is plausible that as energy metabolism shifts, it could trigger
   certain immune responses, either as a direct result of metabolic
   byproducts or as a broader adaptive mechanism to maintain ocular
   homeostasis. Overall, our research underscores the need for a more
   comprehensive approach to understand myopia development, integrating
   insights from energy metabolism, oxidative stress, and immune response
   pathways. Such an integrated perspective could pave the way for more
   targeted therapeutic interventions in the future.

Author Contributions

   Conceptualization, M.S.K. (Man S. Kim) and T.G.K.; methodology, M.S.K.
   (Man S. Kim) and T.G.K.; software, H.K., W.L., Y.-A.K., S.Y. and M.S.K.
   (Man S. Kim); validation, S.Y., J.J., Y.C., Y.L., Y.H.P. and M.S.K.
   (Man S. Kim); formal analysis, S.Y., J.J., Y.C. and Y.L.;
   investigation, H.K., W.L. and M.S.K. (Man S. Kim); resources, H.K.,
   W.L. and M.S.K. (Man S. Kim); data curation, H.K., W.L. and M.S.K. (Man
   S. Kim); writing—original draft preparation, All authors;
   writing—review and editing, M.S.K. (Man S. Kim) and T.G.K.;
   visualization, M.S.K. (Man S. Kim) and T.G.K.; supervision, M.S.K. (Man
   S. Kim) and T.G.K.; project administration, M.S.K. (Man S. Kim) and
   T.G.K.; funding acquisition, T.G.K. All authors have read and agreed to
   the published version of the manuscript.

Institutional Review Board Statement

   The animal study protocol was approved by the animal institutional
   review board of Kyung Hee University hospital at Gangdong (KHNMC AP
   2022-014).

Informed Consent Statement

   Not applicable.

Data Availability Statement

   The data presented in this study are available on request from the
   corresponding author.

Conflicts of Interest

   The authors declare no conflict of interest.

Funding Statement

   This work was supported by a grant from Kyung Hee University in 2022
   (KHU-20220784) and the Korea Health Technology R&D Project through the
   Korea Health Industry Development Institute (KHIDI), funded by the
   Ministry of Health & Welfare, Republic of Korea (grant number:
   HP23C0146).

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References