Development of a BALB/c mice model for blue light retinal damage

Abstract

Blue light exposure can damage the retina, resulting in retinal atrophy and significant vision loss. Currently, no efficient animal models can observe retinal damage caused by blue light within a defined timeframe. Creating a BALB/c mouse model for blue light-induced retinal damage is expected to enhance research focused on the prevention and treatment of age-related macular degeneration. This study explores the potential effect of blue light exposure on the BALB/c mice model by analysing apoptosis and retinal degeneration. Anatomical Pathology Laboratory of Diponegoro University and The Integrated Research and Testing Laboratory of Gadjah Mada University. This study design was a posttest-only control group design. Ten five-week-old BALB/c mice were divided into two groups. The exposure group received 10,000 lux of blue light in the special cage for 2 weeks, 3 h daily. Caspase-3 expression was assessed through polymerase chain reaction testing, and retinal thickness was analyzed using hematoxylin and eosin staining. We used the Shapiro–Wilk test to evaluate data normality. Parametric t-tests and nonparametric Mann–Whitney tests were applied to compare groups, with P < 0.05 considered significant. The average whole retinal thickness of the exposed group was 152.812 ± 20.919 µm, while the control group was 214.948 ± 53.284 µm (P = 0.04). The average caspase-3 expression in the exposed group was 19.03 ± 8.57 µm, while the control group was 5.78 ± 2.63 µm (P = 0.011). This approach, utilizing animal models for blue light exposure, can be employed to learn about retinal damage caused by blue light.

INTRODUCTION

Age-related macular degeneration (AMD) is a significant cause of blindness in individuals aged 50 and above, contributing to 8.7% of global blindness.[1,2] By 2040, it is projected that the global prevalence of AMD will rise by 40% to approximately 288 million people.[3,4,5] In Indonesia, AMD’s prevalence is unknown. Several studies have investigated the correlation between exposure to sunshine and AMD, identifying it as a significant risk factor.[6,7,8]

Blue light is a component of the light spectrum emitted by the sun and has been found to play a significant role in the development of retinal degeneration.[9,10,11] Blue light, with a wavelength ranging from 400 to 500 nm, is emitted by various electronic devices, including computers, televisions, lamps, and cell phones.[12] Blue light can induce oxidative stress, apoptosis, and DNA damage. Excessive blue light exposure leads to oxidative stress and intracellular Reactive Oxygen Species (ROS) generation in the retina.[11,13]

Numerous techniques have been developed. In 2017, Kim et al. conducted a study on the effects of blue light exposure on retinal damage. The study involved 5-week-old BALB/c mice weighing 18–20 g in a standard cage maintained at 25°C ± 1°C temperature. In this study, BALB/c mice were acclimatized for 24 h in a dark room before exposure to blue light. The blue light, with a wavelength of 480 nm and an intensity of 10,000 lux, was administered for 1 h/day in the cage for 14 days. On the final day, the mice could adjust for 24 h in a dark room before being euthanized using a CO₂ chamber.[14]

Currently, no established methods or designated animal models can effectively and conveniently observe damage within a specific timeframe. This research aims, as a preliminary step, to provide a research model to learn the effects of xanthophyll pigments such as lutein and zeaxanthin on AMD more clearly due to the absence of natural pigments in the retina of BALB/c mice.

SUBJECTS AND METHODS

Animals and experimental design

Male BALB/c mice, aged 5 weeks and weighing 18–20 g, were housed in standard cages and provided with unlimited food access. The process involved 2 weeks of acclimatization in a standard cage at 25°C, followed by 24 h of adaptation in a dark environment before treatment. The treatment was conducted in a modified cage equipped with a blue light source emitting at a wavelength of 480 nm, providing a brightness of 10,000 lux [Figure 1]. In addition, mirrors were placed on both sides of the cage. The subjects were exposed to blue light for 3 h daily, from 08.00 to 11.00 WIB, over 2 weeks. A solution of 0.5% tropicamide was administered to the eyes of the mice 30 min beforehand to dilate the pupils and ensure the blue light reached the retina. The mice were carefully positioned in a specialized conical device to keep their eyes fixed on the blue light. After the experiment concluded, the mice were given time to adjust in a dark room for 24 h before being humanely euthanized through cervical dislocation.

Figure 1.

Modified cage. The blue light sources were placed in front of the mice, and mirrors were placed on the other side (a). BALB/c mice were inserted into the remarkable conical (b). The mice’s eyes were fixated toward the blue light (c)

The control group was housed in a standard cage and was not subjected to blue light exposure for the same duration as the treatment group. The animal experiments adhered to ethical guidelines for using animals in ophthalmic and vision research. The protocols complied with the guidelines established by the Anatomical Pathology Laboratory of Diponegoro University and the Integrated Research and Testing Laboratory (LPPT) of Gadjah Mada University. The Health Research Ethics Committee of Diponegoro University approved the animal experimental protocols with No. 10/EC/H/FK-UNDIP/I/2023.

Real-time reverse-transcription polymerase chain reaction caspase-3

The reagent TRIsure was used to isolate total RNA from the retina of mice. The RNA concentration was measured using a NanoDrop 1000 from Thermo Fisher Scientific, Inc. Zymo Research’s Quick-RNA FFPE Kit master mix and SensiFAST^™ SYBR^® No-ROX One-Step Kit were utilized to reverse-transcribe 1 µg of RNA [Figure 2]. Real-time polymerase chain reaction (PCR) was carried out using the SYBR system and caspase-3 primer. The primer sequences used were caspase-3 Forward: 5’ TGGGACTGATGAGGAGA-3’, and caspase-3 Reverse: 5’-ACT GGATGAACCACGAC-3’. The real-time PCR experiment used the StepOnePlus PCR technology.[16]

Figure 2.

Reverse-transcription-polymerase chain reaction examination steps[15]

Histological analysis

After 2 weeks of light exposure, the mice were euthanized by cervical dislocation, and their eyeballs were removed. The eyeballs were fixed in 4% paraformaldehyde overnight at 4°C. Following fixation, the tissue was dehydrated in a graded ethanol series and embedded in paraffin. Thin sections (8–10 µm) were cut from the paraffin blocks, encompassing the optic nerve head to the retinal periphery. These sections were then deparaffinized using xylene and rehydrated through a graded ethanol series. Finally, the sections were stained with hematoxylin and eosin (H and E) to visualize cellular structures for subsequent analysis.[17] The structure difference between retinal BALB/c and C57BL/6 mice lies in the present of pigment in the BALB/c retina [Figure 3]. Quantitative calculations were performed to determine the thickness of the entire retina, achieved by examining six fields of view in each eye, using ×200 magnification and the Olympus CellSens application. The examination results are reported in micrometers.[18]

Figure 3.

Histological sections. (a) BALB/c mice’s eyes and (b) C57BL/6 mice’s eyes[19] GCL: Ganglion Cell Layer, IPL: Inner Plexiform Layer, INL: Inner Nuclear Layer, OPL: Outer Plexiform Layer, ONL: Outer Nuclear Layer, RPE: Retinal Pigment Epithelium, IS: Inner Segments of Photoreceptors, OS: Outer Segments of Photoreceptors

Statistical analyses

Descriptive statistics were calculated for all variables. Normality was assessed using the Shapiro–Wilk test. Differences in caspase-3 expression data were analyzed using the Mann–Whitney test due to abnormal data distribution. In contrast, differences in whole retinal thickness data were assessed with an independent samples t-test because it exhibited normal data distribution. All analyses were performed using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, Illinois, USA) version 29.0.2.0 for macOS, with statistical significance set at P < 0.05.

RESULTS

Retinal histologic alterations induced by light

In the blue light-induced retinal damage model using mice, it was observed that prolonged exposure to blue light had a detrimental impact on the retinal tissue. Our analysis focused on the histologic changes in the retina 2 weeks after light exposure. Figure 4 displays the HE-stained section, which depicts the different retinal layers. In the exposure group, there was a noticeable reduction in the thickness of the whole retina (WR) [Figure 5].

Figure 4.

Whole retina (WR) thickness. (a) WR thickness in the control group. (b) WR thickness in the exposure group. Long-term exposure to blue light significantly reduced the WR thickness

Figure 5.

Blue light affects the retina tissue. The thickness of the whole retina was reduced after 2 weeks of blue light exposure (light exposure group: LE [+], control group: LE [−], n = 5). *The statistical significance is P < 0.05

The statistical data confirmed by the Shapiro–Wilk test for normality (P = 0.22) suggests that the data was the normal distribution. There was a noticeable disparity in the average thickness of the WR between the control group and the light exposure group [Figure 5] (214.948 ± 53.248 µm and 152.812 ± 20.919 µm; Independent t-test P = 0.04*). This difference was statistically significant (P < 0.05), indicating a 62.136 μm reduction in retinal thickness in the light exposure group compared to the control group after 2 weeks of blue light exposure.

Light exposure enhanced caspase-3 expression

The expression of caspase-3 in the retina showed a significant increase 2 weeks after exposure to blue light, as depicted in Figure 6. There was a noticeable disparity in the mean caspase-3 expression between the control and exposure groups (5.78 ± 2.64 and 19.03 ± 8.58, respectively; Mann–Whitney test P = 0.011).

Figure 6.

Caspase-3 accumulation increased in response to blue light exposure. Expression of caspase-3 increases in the light exposure group compared to the control group. Mann–Whitney test P = 0.011* (Light exposure group: LE [+], control group: LE [−])

DISCUSSION

Blue light is part of the sun’s spectrum and has been linked to retinal degeneration.[9,10] Exposure to blue light is regarded as a risk factor for the onset of AMD degeneration.[15,20] Prolonged exposure to light can cause damage to the cells that are found in the retina. When the electron returns to its original state, it releases the additional energy, initiating a molecular exchange that results in the creation of ROS. Light can have a detrimental effect on the photoreceptor due to photooxidative reactions. Exposure to certain types of light, especially the blue spectrum, has been found to affect the health of the photoreceptors negatively.

Blue light can elevate intracellular ROS and cause oxidative damage. Excessive ROS can trigger the fragmentation of mitochondria by upregulating the expression of dynamin-related protein 1 and downregulating the expression of mitofusin-2.[21] In addition, blue light can interfere with the calcium homeostasis of mitochondria, reduce the potential across the membrane, and increase the permeability of the mitochondrial membrane. The release of Cytochrome C and Apoptotic protease activating factor-1 (Apaf-1) from mitochondria into the cytoplasm triggers the activation of caspase-3, caspase-6, and caspase-7 through a series of events known as the caspase cascade. Activated caspase proteins are crucial in cell apoptosis.[20,22]

The BALB/c mice are also commonly used in vision research. The BALB/c mouse has been utilized to investigate various human diseases, such as AMD, aiming to explore the benefits of light preconditioning in reducing oxidative stress and the inflammatory responses triggered by phototoxicity through cytokine mediation.[23]

In a recent study by Miralles de Imperial-Ollero et al. and a previous study by Kim et al., BALB/c mice were used to provide an eye damage induction effect at blue light wavelengths of 460 ± 10 nm with an exposure duration of 2 h continuously for 5 days. BALB/c mice had representative results using blue light intensities of 1000, 2000, and 3000 lux and exposure durations of 24, 48, and 72 h.[21,24]

Another method used a Wistar mouse model by providing acute and chronic/long-term exposure to wavelengths of white light, blue light 460 ± 5 nm, and green light 530 ± 10 nm. Acute exposure is given light constantly for 6, 12, 18, 24, 48, or 72 h with light intensities of 102, 234, 268, and 500 lux.[21,25] Long-term/chronic exposure is given by the 12-h On/Off cycle method for 8 or 28 days using a light intensity of 500 lux.[21,26]

Staining with H and E and analysis of caspase-3 expression revealed that prolonged exposure to blue light led to apoptosis and cell death throughout the retina. Over 2 weeks, exposure to blue light resulted in a noticeable reduction in the thickness of the entire retina of retinal tissue. This observation was made by carefully examining six fields of view in each eye. The results are visually represented in Figures 5 and 6, providing clear evidence of the impact of blue light exposure.

After exposure to blue light, the BALB/c mouse strain exhibited elevated caspase-3 levels. The expression of caspase-3 was found to be higher in the light exposure group than in the control group. This difference in expression levels can be attributed to the apoptosis process, which reduced the retinal thickness. Caspase-3 plays a crucial role in apoptotic cell death, being activated through cleavage.

This study has limitations, such as the need for more analysis of varying light-intensity doses and evaluation of optimal exposure times. Another limitation is that it only studied caspase-3 as the executor of apoptosis without examining other oxidative stress markers, such as superoxide, dismutase, and catalase.

CONCLUSION

The findings of this study demonstrated that the proposed method effectively created a mouse model of blue light-induced retinal damage, and it can serve as a viable option for further research on the effects of blue light.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil