August 2024
Volume 65, Issue 10
Open Access
Retina  |   August 2024
Effects of Acute High-Altitude Exposure on Morphology and Function of Retinal Ganglion Cell in Mice
Author Affiliations & Notes
  • Yi Yang
    Department of Ophthalmology, the Second Hospital of Lanzhou University, Lanzhou, Gansu, People's Republic of China
  • Cong Han
    Department of Ophthalmology, the Second Hospital of Lanzhou University, Lanzhou, Gansu, People's Republic of China
  • Yi Sun
    Department of Ophthalmology, the 940th Hospital of Joint Service Support Forces of the Chinese People's Liberation Army, Lanzhou, Gansu, People's Republic of China
  • Xin Zhao
    Department of Ophthalmology, the Second Hospital of Lanzhou University, Lanzhou, Gansu, People's Republic of China
  • Zhaoqian Chen
    Department of Ophthalmology, the Second Hospital of Lanzhou University, Lanzhou, Gansu, People's Republic of China
  • Liangtao Zhao
    Cuiying Biomedical Research Center, the Second Hospital of Lanzhou University, Lanzhou, Gansu, People's Republic of China
  • Yuting Li
    Department of Pathology, Basic Medical School, Ningxia Medical University, Yinchuan, China
  • Wenfang Zhang
    Department of Ophthalmology, the Second Hospital of Lanzhou University, Lanzhou, Gansu, People's Republic of China
  • Correspondences: Wenfang Zhang, Department of Ophthalmology, The Second Hospital of Lanzhou University, Lanzhou, Gansu 730000, People's Republic of China; [email protected]
  • Yuting Li, Department of Pathology, Basic Medical School, Ningxia Medical University, Yinchuan 750004, China; [email protected]
  • Footnotes
     YY and CH contributed equally to this work.
Investigative Ophthalmology & Visual Science August 2024, Vol.65, 19. doi:https://doi.org/10.1167/iovs.65.10.19
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      Yi Yang, Cong Han, Yi Sun, Xin Zhao, Zhaoqian Chen, Liangtao Zhao, Yuting Li, Wenfang Zhang; Effects of Acute High-Altitude Exposure on Morphology and Function of Retinal Ganglion Cell in Mice. Invest. Ophthalmol. Vis. Sci. 2024;65(10):19. https://doi.org/10.1167/iovs.65.10.19.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: High altitude retinopathy (HAR) is a retinal functional disorder caused by inadequate adaptation after exposure to high altitude. However, the cellular and molecular mechanisms underlying retinal dysfunction remain elusive. Retinal ganglion cell (RGC) injury is the most important pathological basis for most retinal and optic nerve diseases. Studies focusing on RGC injury after high-altitude exposure (HAE) are scanty. Therefore, the present study sought to explore both functional and morphological alterations of RGCs after HAE.

Methods: A mouse model of acute hypobaric hypoxia was established by mimicking the conditions of a high altitude of 5000 m. After HAE for 2, 4, 6, 10, 24, and 72 hours, the functional and morphological alterations of RGCs were assessed using retinal hematoxylin and eosin (H&E) sections, retinal whole mounts, transmission electron microscopy (TEM), and the photopic negative response (PhNR) of the electroretinogram.

Results: Compared with the control group, the thickness of the ganglion cell layer and retinal nerve fiber layer increased significantly, RGC loss remained significant, and the amplitudes of a-wave, b-wave, and PhNR were significantly reduced after HAE. In addition, RGCs and their axons exhibited an abnormal ultrastructure after HAE, including nuclear membrane abnormalities, uneven distribution of chromatin in the nucleus, decreased cytoplasmic electron density, widening and vacuolization of the gap between axons, loosening and disorder of myelin sheath structure, widening of the gap between myelin sheath and axon membrane, decreased axoplasmic density, unclear microtubule and nerve fiber structure, and abnormal mitochondrial structure (mostly swollen, with widened membrane gaps and reduced cristae and vacuolization).

Conclusions: The study findings confirm that the morphology and function of RGCs are damaged after HAE. These findings lay the foundation for further study of the specific molecular mechanisms of HAR and promote the effective prevention.

High altitude retinopathy (HAR) is a retinal dysfunction disease associated with hypoxia and low barometric pressure, often occurring in individuals who rapidly ascend to high altitude areas with inadequate adaptation.1 The most common clinical manifestations of HAR include retinal hemorrhage, retinal vessel dilation and curvature, optic disc congestion, and swelling.2 However, macular edema,3 retinal artery4 or vein5 occlusion, and vitreous hemorrhage6 are rare clinical manifestations of this disorder. With the rapid development of transportation and tourism, individuals in low altitude areas can quickly enter high altitude areas without enough time to adapt, leading to an increase in the incidence rate of HAR annually.7 Currently, effective methods for preventing and treating HAR are limited due to the lack of a comprehensive understanding of the pathogenesis of HAR. 
Retinal ganglion cells (RGCs) — with cell bodies located in the innermost layer of the retina and their axons forming the optic nerve — are the only output neurons that transmit visual signals from the eyes to the brain.8 Functional and/or morphological alterations of RGCs often lead to visual impairment or even irreversible blindness. Increasing evidence suggests that RGCs are susceptible to various types of injuries, such as ischemia,9 oxidative stress,10 mitochondrial dysfunction,11 excitotoxic damage glutamate excitotoxicity,12 and glial cell activation.13 However, only a handful of studies have reported on the functions and morphology of RGCs exposed to high altitude and low pressure. 
Herein, a mouse model of acute hypobaric hypoxia (AHH) was developed by simulating the conditions of a high altitude of 5000 m. Functional and morphological alterations of RGCs were analyzed at different time points after exposure to high altitudes using light microscopy, electron microscopy, and the photopic negative response (PhNR) of the electroretinogram. The study findings will provide novel insights into the pathophysiology of HAR. 
Materials and Methods
Animals
Adult male C57BL/6 mice (7–8 weeks old, weighing 20 ± 5 g, Certificate number: SCXK Gansu 2020-0002) were purchased from the Lanzhou Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences (Lanzhou, Gansu, China). Before the trial, the mice were housed in standard cages in an animal room (room temperature = 23 ± 2°C; light/dark cycle = 12 hours/12 hours; relative humidity = 60%; humidity = 40–50%) for 1 week, with food and water ad libitum. All animal procedures were performed per the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. This study was approved by the Animal Ethics Committee of Lanzhou University Second Hospital (Approval number: D2019-111). 
Establishment of the Animal Model of AHH
After adaptive feeding for 1 week, the experimental group of mice were sequentially housed in the high-altitude negative pressure simulation cabin for 2, 4, 6, 10, 24, and 72 hours. The control system parameters for the high-altitude experimental simulation cabin were set to simulate an altitude of 5000 m, with an oxygen partial pressure of 11.3 kPa, air pressure of 54.02 kPa, temperature of 25 ± 2°C, relative humidity of 40 ± 5%, simulated altitude rise speed of 14.6 m/s, SP1 of 0.304, and SP2 of 0.307. The high-altitude experimental simulation cabin maintained a stable pressure inside and a 12/12-hour light/dark cycle. Food and water were provided ad libitum in the high-altitude negative pressure simulation cabin. No accidental mortality was observed among the mice during high-altitude simulation in the negative pressure chamber. 
Hematoxylin and Eosin Staining
Mice were immediately euthanized after leaving the high-altitude negative pressure simulation cabin. Their eyes were enucleated and fixed in 4% paraformaldehyde (PFA) for 24 hours at room temperature. Subsequently, the eyes were dehydrated in ethanol solution and embedded in paraffin. Specimens were cut into 3-µm thick retinal cross-sections using a cryostat (Leica RM2235; Leica, Germany). Three slices were prepared for each eye and stained with hematoxylin and eosin (H&E), the stained retinal sections were photographed using the TissueFAXS PLUS Scanning System (TissueGnostics Asia Pacific Ltd., TissueGnostics, Austria). Twenty points were selected equidistant from the posterior pole of the retina to the peripheral region on the micrograph of the retina slice (magnification 200times; the starting point was the junction of the optic nerve and retinal pigment epithelium, and the distance between the points was 25 µm; Fig. 1A). The thicknesses of the retinal nerve fiber layer (RNFL) and retinal ganglion cell layer (GCL) were measured at 20 points on the left and 20 points on the right side of the optic disc on the retinal slice and averaged to obtain the values for one eye. Five mice were measured in each group to obtain representative data. 
Figure 1.
 
The thickness of the GCL and RNFL was increased with acute high-altitude exposed time prolonged. (A) Representative H&E-staining images of mouse retina (scale = 100 µm). Briefly, 20 points were selected equidistant from the posterior pole of the retina toward the peripheral region on the micrograph of the retina slice magnified at 200 times (the starting point is the junction of the optic nerve and retinal pigment epithelium, and the distance between the points is 25 µm). The thickness of the nerve fiber layer (RNFL) and retinal ganglion cell layer (GCL) was assessed at 20 locations on both the left and right sides of the optic disc on retinal slices. The measurements from each eye were averaged to obtain the final values. To collect representative data, five mice were recorded in each group. (B and C) Representative regions of H&E-stained retinal cross sections of the control and acute high-altitude exposed for 2, 4, 6, 10, 24, and 72 hours. Images were captured at the same magnification. Scale bar = 100 µm. (D) Calculation of thickness of the GCL and RNFL in the control group and high-altitude exposed groups at 2, 4, 6, 10, 24, and 72 hours. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group.
Figure 1.
 
The thickness of the GCL and RNFL was increased with acute high-altitude exposed time prolonged. (A) Representative H&E-staining images of mouse retina (scale = 100 µm). Briefly, 20 points were selected equidistant from the posterior pole of the retina toward the peripheral region on the micrograph of the retina slice magnified at 200 times (the starting point is the junction of the optic nerve and retinal pigment epithelium, and the distance between the points is 25 µm). The thickness of the nerve fiber layer (RNFL) and retinal ganglion cell layer (GCL) was assessed at 20 locations on both the left and right sides of the optic disc on retinal slices. The measurements from each eye were averaged to obtain the final values. To collect representative data, five mice were recorded in each group. (B and C) Representative regions of H&E-stained retinal cross sections of the control and acute high-altitude exposed for 2, 4, 6, 10, 24, and 72 hours. Images were captured at the same magnification. Scale bar = 100 µm. (D) Calculation of thickness of the GCL and RNFL in the control group and high-altitude exposed groups at 2, 4, 6, 10, 24, and 72 hours. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group.
Retinal Whole Mounts for Antibody Staining and RGC Counting
After euthanizing the mice, their eyeballs were quickly removed and fixed in a 4% PFA solution for 30 minutes. The cornea, iris, and lens were dissected away from each eyecup. The retina was peeled off the eyecup, cut into four uniform pieces, and laid flat on a glass slide. The fixed retinas were washed thrice in 0.01 M phosphate-buffered saline (PBS) for 5 minutes each time and blocked in 10% normal donkey serum for 1 hour. Then, the retinas were incubated in the anti-Brn3a antibody solution (dilution 1:100, Santa Cruz, sc-8429) for 24 hours. The retinas were washed thrice using PBS for 5 minutes each time. Next, retinas were incubated in a secondary antibody solution (Donkey Anti-Mouse IgG H&L, Alexa Fluor 594, Jackson Immuno Research Laboratories, Inc., USA) at room temperature for 2 hours. The retinas were rinsed thrice with PBS for 5 minutes each time. Finally, the retinas were dripped with anti-fluorescence quenching and sealing agent containing 4′,6-diamino-2-phenylindole (DAPI; SouthBiotech, USA), and then covered with a cover glass slide for sealing. Images were obtained using the TissueFAXS PLUS Scanning System (TissueGnostics Asia Pacific Ltd., TissueGnostics, Austria). 
Eight images (magnification 200×) were obtained from the peripheral and central regions of each retina's four quadrants. The number of cells in each image was counted in a masked manner, and the average cell count for the four peripheral and four central images was calculated. The number of RGCs in three mice per group was calculated. 
Transmission Electron Microscopy
After fixing the enucleated eyes in a mixed solution of 4% PFA and 2.5% glutaraldehyde for 24 hours, the cornea, iris, and lens were dissected away from each eyecup. Then, the retinal choroid sclera complex/retrobulbar optic nerves were cut into 1-mm3 tissue blocks and were fixed for 2 hours. After softening with 3% ethylenediaminetetraacetic acid disodium (EDTA Na2) for 20 minutes, blocks of the retina/optic nerve tissue were fixed again in a 1% osmium acid solution for 2 hours. Next, the samples were dehydrated with graded alcohol and embedded in epoxy resin. Subsequently, the sample was sliced into ultra-thin sections with a thickness of 70 nm using an ultra-thin slicer and then observed and photographed under HT7800 transmission electron microscopy (TEM; JEOL, Tokyo, Japan). 
Measurement of the PhNR
The PhNR of mice was measured immediately after leaving the high-altitude negative pressure simulation cabin. After the pupils of the mice were dilated with compound tropicamide eye drops, the mice were fully anesthetized by intraperitoneal injection of tribromoethanol. The mice were placed on a heat pad fixed on the ERG stage to maintain its body temperature at 37°C. Recording electrodes with wire loops were placed in the center of the mouse's cornea that had been pre-coated with carbomer eye drops. The ground electrode was inserted into the mouse's tail, and the reference electrode was inserted into the subcutaneous area of both cheeks. Subsequently, after adapting to a white light background for 10 minutes, measurements were initiated using the PhNR testing program of the Ganzfeld visual electrophysiology system (Q450 SCX Ganzfeld, Roland Consult, Brandenburg, Germany). The stimulus frequency was 2 hertz (Hz), and white light with an intensity of 10 candelas per square meter (cd/m2) was presented for 4 milliseconds against a white background (30 cd/m2). Three waveforms were measured for each eye of each mouse and the results were averaged. The mice were adapted to a blue background (20 cds/m2) for 10 minutes, followed by red light stimulation (3 cd/m2). Each eye of the mouse was tested at least three times, and the average of the three tests was taken. Four mice per group were measured. The PhNR amplitude was defined as the difference between the baseline and the peak of the negative wave following the b-wave. The amplitudes of PhNR were then compared among the groups. The average implicit time of PhNR was 123.26 ± 6.59 ms, which was within the range of times previously reported for the rodent PhNR.14,15 
Statistical Analysis
All data were analyzed and visualized with SPSS 24.0 software and GraphPad Prism 8.0 Software. Data were expressed as mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post hoc test was used to analyze and compare differences among groups. A P value of < 0.05 was considered statistically significant. The asterisks in all figures indicate the degree of significant difference compared with the control group (*P < 0.05; * *P < 0.01; * * *P < 0.001; and * * * *P < 0.0001). 
Results
Analysis of GCL and RNFL Thickness After Acute High-Altitude Exposure
To evaluate the morphological effects of acute HAE on RGCs, GCL, and RNFL the thicknesses were measured after H&E staining. As shown in Figure 1B, the GCL and RNFL displayed a normal structure in the control group, with a mean thickness of 26.28 ± 0.81 µm. Contrastingly, H&E staining revealed that the structures of the GCL and RNFL were disordered after acute HAE (Fig. 1C). The mean thickness of the GCL and RNFL after acute HAE for 2, 4, 6, 10, 24, and 72 hours were 29.58 ± 0.91, 30.72 ± 1.11, 32.97 ± 1.54, 34.23 ± 1.17, 33.85 ± 0.91, and 35.92 ± 1.21 µm, respectively. Compared with the control group, the thickness of the GCL and RNFL was markedly increased after 6, 10, 24, and 72 hours of acute HAE (P < 0.01, P < 0.0001, P < 0.001, and P < 0.0001, respectively; Fig. 1D). 
Quantification of RGCs After Acute HAE
RGC loss with increasing HAE time was evaluated by analyzing the RGC density of the control and HAE groups after 2, 4, 6, 10, 24, and 72 hours. As shown in Figures 2A and 2B, the RGC density with HAE time in the central and peripheral retina was assessed by counting the number of RGCs using immunofluorescence staining with Brn3a in flat-mounted retinas. The mean densities of central and peripheral RGCs in the control group were 3405.69 ± 216.03 cells/mm2 and 2776.46 ± 200.46 cells/mm2, respectively. Meanwhile, the mean densities of central and peripheral RGCs were 3450.72 ± 84.76 and 2865.33 ± 216.86, 3211.35 ± 70.78 and 2605.82 ± 58.95, 3310.89 ± 122.62 and 2358.15 ± 300.99, 3009.31 ± 93.53 and 2259.20 ± 109.74, 2973.76 ± 99.76 and 2328.53 ± 207.87, and 2654.40 ± 186.24 and 1765.65 ± 194.51 cells/mm2 after 2, 4, 6, 10, 24, and 72 hours of HAE, respectively. No difference in central and peripheral RGC densities was observed between the control and HAE groups after 2, 4, 6, 10, and 24 hours of HAE; however, the RGC loss in the center and periphery of the retina was statistically significant after 72 hours of HAE, compared with the control group (Figs. 2C, 2D). 
Figure 2.
 
The loss of RGCs in both the central and peripheral regions of the retina increases with prolonged exposure to high altitude. (A) Whole-mount retinas were immunostained with Brn3a and RGCs were quantified in two sampling areas (at 500 µm from the optic nerve head for the center and 500 µm from the peripheral edge for the periphery) in each of the four quadrants of the retina. Scale bar = 500 µm. (B) Representative high-magnification images of BRN3A immunostaining RGCs from the central or peripheral retina at the control group and high-altitude exposure for 2, 4, 6, 10, 24, and 72 hours. Scale bar = 100 µm. (C) Quantitative analysis of BRN3A+ immunostaining RGCs density in the retinal center. (D) Quantitative analysis of BRN3A+ immunostaining RGCs density in the retinal periphery. Data are the mean ± SEM; * P < 0.05 versus the control.
Figure 2.
 
The loss of RGCs in both the central and peripheral regions of the retina increases with prolonged exposure to high altitude. (A) Whole-mount retinas were immunostained with Brn3a and RGCs were quantified in two sampling areas (at 500 µm from the optic nerve head for the center and 500 µm from the peripheral edge for the periphery) in each of the four quadrants of the retina. Scale bar = 500 µm. (B) Representative high-magnification images of BRN3A immunostaining RGCs from the central or peripheral retina at the control group and high-altitude exposure for 2, 4, 6, 10, 24, and 72 hours. Scale bar = 100 µm. (C) Quantitative analysis of BRN3A+ immunostaining RGCs density in the retinal center. (D) Quantitative analysis of BRN3A+ immunostaining RGCs density in the retinal periphery. Data are the mean ± SEM; * P < 0.05 versus the control.
Analysis of Ultrastructural Changes of RGCs and Their Axons After Acute HAE Under TEM
Next, TEM was applied to assess ultrastructural changes in RGCs and their axons 2 to 72 hours after HAE to further understand the lesions of RGCs after acute HAE. As shown in Figure 3, RGCs exhibited normal ultrastructures with intact cell structure, smooth nuclear membrane, uniformly distributed chromatin, moderate electron density in the cytoplasm, and a clear mitochondrial structure in the control group. Conversely, RGCs exhibited abnormal cellular structures with an increase in the exposure time in the HAE group, including nuclear membrane abnormalities (bending, widening, invagination, endometrial stratification, and formation of outer membrane protrusions), uneven distribution of chromatin in the nucleus, decreased cytoplasmic electron density, disordered mitochondrial structure with increased volume, widened membrane gaps, and reduced cristae. More double or multi-membrane autophagic vesicles containing cell organelles were observed in the dendrites and cytoplasm of RGCs after 4 hours of HAE. Some mitochondria showed a significant volume reduction and an increased mitochondrial membrane electron density after 72 hours of HAE. 
Figure 3.
 
The ultrastructural images of retinal RGCs from C57BL/6 mice. Normal ultrastructure of RGCs with intact cell structure, smooth nuclear membrane, uniform distributed chromatin, moderate electron density in the cytoplasm, and clear mitochondrial structure in the control group. RGCs exhibited abnormal cellular structure, including nuclear membrane abnormalities, uneven distribution of chromatin in the nucleus, decreased cytoplasmic electron density, disordered mitochondrial structure with increased volume, widened membrane gaps, and reduced cristae at 2 to72 hours after high-altitude exposure. Au = autophagosome; M = mitochondria. Scale bar = 2 µm (upper panel) and 1 µm (lower panel).
Figure 3.
 
The ultrastructural images of retinal RGCs from C57BL/6 mice. Normal ultrastructure of RGCs with intact cell structure, smooth nuclear membrane, uniform distributed chromatin, moderate electron density in the cytoplasm, and clear mitochondrial structure in the control group. RGCs exhibited abnormal cellular structure, including nuclear membrane abnormalities, uneven distribution of chromatin in the nucleus, decreased cytoplasmic electron density, disordered mitochondrial structure with increased volume, widened membrane gaps, and reduced cristae at 2 to72 hours after high-altitude exposure. Au = autophagosome; M = mitochondria. Scale bar = 2 µm (upper panel) and 1 µm (lower panel).
Ultrastructural changes in RGC axons were also observed via TEM after HAE. TEM results revealed a normal ultrastructure of RGC axons in the control group, with a clear hierarchy, dense and regular arrangement of myelin sheaths, tightly connected myelin sheath and axial membrane, well-arranged microtubules and nerve fibers, and normal mitochondrial structure. However, RGC axons exhibited abnormal ultrastructures with an increase in the exposure time in the HAE group, including widening and vacuolization of the gap between axons, loosening and disorder of myelin sheath structure, widening of the gap between the myelin sheath and the axon membrane, decreased axoplasmic density, unclear microtubule and nerve fiber structure, and abnormal mitochondrial structure (mostly swollen, with reduced cristae and vacuolization; Fig. 4). Ultrastructural changes of the above-mentioned axons were most pronounced after 10 hours of HAE. More autophagosomes were observed in axons after 24 hours of HAE. However, the ultrastructure of the axons showed opposite changes after 72 hours of HAE, with a dense myelin sheath arrangement and increased electron density, reduced fibrotic gaps between axons, decreased and concentrated volume of mitochondria inside the axons, and increased electron density. 
Figure 4.
 
Ultrastructural images of axons in RGCs from C57BL/6 mice. Normal ultrastructure of axons in RGCs of the control group, with clear hierarchy, dense and regular arrangement of myelin sheaths, tightly connected myelin sheath and axial membrane, well arranged microtubules and nerve fibers, and normal structure mitochondria. The ultrastructure of axons within RGCs displayed abnormalities between 2 and 72 hours following high-altitude exposure. These abnormalities included widened and vacuolized gaps between axons, loosening and disorganization of the myelin sheath structure, widening of the gap between the myelin sheath and axon membrane, reduced axoplasmic density, indistinct microtubule and nerve fiber structure, and irregular mitochondrial morphology (mostly swollen, with reduced cristae and vacuolization). Au = autophagosome; M = mitochondria. Scale bar = 1 µm (upper panel) and 500 nm (lower panel).
Figure 4.
 
Ultrastructural images of axons in RGCs from C57BL/6 mice. Normal ultrastructure of axons in RGCs of the control group, with clear hierarchy, dense and regular arrangement of myelin sheaths, tightly connected myelin sheath and axial membrane, well arranged microtubules and nerve fibers, and normal structure mitochondria. The ultrastructure of axons within RGCs displayed abnormalities between 2 and 72 hours following high-altitude exposure. These abnormalities included widened and vacuolized gaps between axons, loosening and disorganization of the myelin sheath structure, widening of the gap between the myelin sheath and axon membrane, reduced axoplasmic density, indistinct microtubule and nerve fiber structure, and irregular mitochondrial morphology (mostly swollen, with reduced cristae and vacuolization). Au = autophagosome; M = mitochondria. Scale bar = 1 µm (upper panel) and 500 nm (lower panel).
Evaluation of Retinal Functions by ERG After Acute HAE
We evaluated the effects of acute HAE on RGC function using PhNR recorded under two different stimulation conditions (white stimuli on a white background [W/W] and red stimuli on a blue background [R/B]). Figures 5A and 5B show representative PhNR elicited by the W/W and R/B stimuli in the control group and the HAE group with different exposure times. In the control group, the mean amplitudes of a-wave, b-wave, and PhNR elicited by W/W and R/B stimuli were 24.15 ± 1.33, 111.08 ± 4.32, and 30.34 ± 1.39 µV and 9.17 ± 1.25, 17.37 ± 2.59, and 7.31 ± 0.54 µV, respectively. The amplitude of PhNR elicited by W/W stimuli was larger than that elicited by R/B stimuli. The average amplitudes of a-wave, b-wave, and PhNR elicited by W/W stimuli after 2, 4, 6, 10, 24, and 72 hours of HAE were 18.59 ± 2.31, 86.18 ± 10.06, and 18.29 ± 1.59; 16.89 ± 1.54, 83.64 ± 7.29, and 15.39 ± 0.90; 17.42 ± 1.82, 77.70 ± 4.28, and 13.35 ± 1.02; 17.07 ± 0.78, 81.72 ± 8.06, and 15.64 ± 0.12; 18.06 ± 2.02, 85.60 ± 6.81, and 15.56 ± 1.93; and 16.75 ± 1.74, 79.71 ± 10.49, and 12.79 ± 1.83 µV, respectively. The amplitude of PhNR began to significantly decrease from 2 hours of HAE, reaching its lowest value at 6 hours. There was an increasing trend at 10 and 24 hours compared with 6 hours, followed by a decrease at 72 hours. However, compared with the control group, the reduction in the PhNR amplitude in each HAE time point was statistically significant (P < 0.01; Fig. 5C). The average amplitudes of a-wave, b-wave, and PhNR elicited by R/B stimuli after 2, 4, 6, 10, 24, and 72 hours of HAE were 5.72 ± 0.29, 12.75 ± 2.09, and 5.53 ± 0.28; 4.95 ± 0.82, 10.19 ± 1.64, and 3.61 ± 0.25; 5.81 ± 0.79, 10.88 ± 1.34, and 2.71 ± 0.23; 6.03 ± 0.69, 10.05 ± 1.54, and 3.75 ± 0.45; 6.50 ± 0.76, 10.83 ± 0.68, and 3.96 ± 0.56; and 5.49 ± 0.84,10.85 ± 1.54, and 2.54 ± 0.27 µV, respectively. The amplitude of PhNR decreased significantly with an increase in the exposure time at high altitudes, reaching its lowest point at 6 hours. There was an increasing trend at 10 and 24 hours compared with 6 hours, followed by a decrease at 72 hours. Except for 2 hours, the amplitude of PhNR decreased significantly at various time points after HAE compared with the control group (P < 0.01; Fig. 5D). 
Figure 5.
 
(A and B) Representative PhNR elicited by the W/W and R/B stimuli in the control group and high-altitude exposure group with different exposure times. (C) The amplitudes of a-wave, b-wave, and PhNR elicited by W/W stimuli in the RGCs at control and at each time point after high-altitude exposure (2 hours, 4 hours, 6 hours, 10 hours, 24 hours, and 72 hours) are compared. When compared to the control group, the reduction of PhNR amplitude in each high-altitude exposure time point was statistically significant. Data are presented as the mean ± SEM; ***P < 0.001 and ****P < 0.0001 versus the control. (D) The amplitudes of a-wave, b-wave, and PhNR elicited by R/B stimuli in the RGCs at control and at each time point after high-altitude exposure (2 hours, 4 hours, 6 hours, 10 hours, 24 hours, and 72 hours) are compared. The amplitude of PhNR significantly decreased compared to the control group at various time points after high-altitude exposure except for 2 hours. Data are presented as the mean ± SEM; **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus the control group.
Figure 5.
 
(A and B) Representative PhNR elicited by the W/W and R/B stimuli in the control group and high-altitude exposure group with different exposure times. (C) The amplitudes of a-wave, b-wave, and PhNR elicited by W/W stimuli in the RGCs at control and at each time point after high-altitude exposure (2 hours, 4 hours, 6 hours, 10 hours, 24 hours, and 72 hours) are compared. When compared to the control group, the reduction of PhNR amplitude in each high-altitude exposure time point was statistically significant. Data are presented as the mean ± SEM; ***P < 0.001 and ****P < 0.0001 versus the control. (D) The amplitudes of a-wave, b-wave, and PhNR elicited by R/B stimuli in the RGCs at control and at each time point after high-altitude exposure (2 hours, 4 hours, 6 hours, 10 hours, 24 hours, and 72 hours) are compared. The amplitude of PhNR significantly decreased compared to the control group at various time points after high-altitude exposure except for 2 hours. Data are presented as the mean ± SEM; **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus the control group.
Discussion
The present study analyzed the effects of acute HAE on the thickness of GCLs and RNFLs, density of RGCs, ultrastructure of RGCs and their axons, and RGC function through experimental observation in a mouse model of AHH. The findings confirmed that RGCs were significantly damaged in both function and morphology after acute HAE. 
RGCs are the only pathway for the brain to access the visual world. Their cell bodies and axons form the innermost layer of the retina (RNFL and GCL), where the nerve fiber layer converges to form the optic nerve, transmitting visual information to the brain. RGC injury is the most important pathological basis for visual impairment caused by various retinal and optic nerve diseases. Therefore, observing the morphological or functional changes in RGCs and their axons is a key indicator for understanding the progression of HAR. 
In recent years, some studies have reported a correlation between RNFL thickness and high-altitude retinopathy.16 For example, Tian et al.17 found that the thickness of RNFL and GCL increased significantly after rapid exposure to a high-altitude environment in Tibet. Yin et al.18 quantitatively analyzed changes in the retinal structure of 109 healthy subjects during acute exposure to high altitude (3700 m) using spectral domain optical coherence tomography (OCT) and found that high-altitude environments increased the RNFL thickness and macular thickness. Ma et al.19 also found that compared with healthy high-altitude residents, high-altitude residents with high-altitude polycythemia had a significant increase in RNFL thickness. The current study also confirmed that mouse retinal nerve fiber (RNF) and GCL exhibited varying degrees of thickening after acute HAE, consistent with the aforementioned studies. So far, although the exact pathological and physiological mechanisms underlying acute exposure to high-altitude areas leading to retinal RNFL and GCL thickening remain elusive, numerous studies have shown that altitude-related hypoxia may be the main factor causing changes in RNFL and GCL thickness. First, the retina is one of the most metabolically active tissues in the human body. It has two blood circulation systems, namely the choroidal circulation system, which supplies the outer layer of the retina, and the retinal circulation system, which supplies the inner layer of the retina. The retinal blood flow is strictly regulated by tissue oxygen tension (PO2). A decrease in the arterial partial pressure of oxygen (PaO2) immediately causes an increase in retinal blood flow.20,21 The superficial capillary network of retinal blood vessels is mainly distributed in the GCL of the retina. In addition, the capillary plexus around the radial papilla is located in the RNFL. Therefore, altitude-related hypoxia-induced vasodilation can particularly lead to RNFL and GCL thickening. A study on long-term low-pressure hypoxia in mountaineers at the highest altitude of 5300 meters reported a significant increase in retinal blood flow. They found that the retinal blood flow increased by 89% within 2 hours of reaching the altitude, increased by 128% after 5 days compared with the control blood flow, and increased by 174% after 7 weeks.21 Yang et al. also confirmed that short-term exposure to high altitude can cause a significant increase in the retinal vein diameter.22 Second, hypobaric hypoxia has been found to directly damage the tight junctions of retinal endothelial cells by stimulating the production and release of inflammatory factors,23,24 mediating oxidative stress,25 damaging the inner blood-retinal barrier, and causing vascular retinal edema. In addition, previous studies have shown that RGCs are particularly sensitive to acute and transient hypoxic stress. Hypoxia-induced activation of nitric oxide synthase (NOS), excessive release of glutamate, and increased intracellular calcium ion can lead to mitochondrial dysfunction in RGCs, resulting in toxic edema and death of RGCs, which are the main factors associated with RGC loss.26,27 Meanwhile, RGC swelling is also considered a component of retinal edema. We also confirmed through TEM analysis of the ultrastructure of RGCs that cell bodies, axons, and organelles (such as mitochondria) of RGCs undergo edema after acute HAE. Additionally, retinal H&E staining showed that the RNF and GCL thicknesses increased with exposure time in mice exposed to 5000 m altitude conditions and were highest at 10 hours of hypoxia exposure, decreased at 24 hours, and then increased again at 72 hours. We believe that the above trend of changes is the result of a series of adaptive adjustments made by the body to cope with the challenge of low-pressure hypoxia. Although there is currently a general understanding of systemic changes associated with adaptation to HAE, the potential molecular and cellular processes have not been fully elucidated and warrant further research. 
Currently, hypoxia is considered to play a crucial role in the pathophysiology of multiple common retinal diseases, many studies2832 have focused on the effects of hypoxia on retinal function (such as changes in various components of electroretinogram). However, to the best of our knowledge, no studies have examined RGC function in animals with histopathologically confirmed high-altitude hypoxia-induced RGC injuries. Nonetheless, only a few methods to quantify RGC function are available at present. PhNR is a slow negative component of the full-field electroretinogram that occurs after the b-wave and has been shown to objectively evaluate the generalized activity of the axon and soma of RGCs.33 Although visual evoked potentials (VEPs), pattern electroretinograms (PERGs), and scotopic threshold response can also objectively detect RGC function, PhNR waves may have some practical advantages over these techniques. Compared to traditional methods, PhNR offers significant advantages for assessing RGC function. It enables faster and easier measurement without the need for long-term dark adaptation, refraction correction, or being influenced by the clarity of the refractive media.33 Accumulating studies have proven that PhNR is an objective tool for detecting RGC dysfunction in diseases such as glaucoma, optic neuropathy, anterior ischemic optic neuropathy, tumor-induced compressive optic neuropathy, and retinal vein occlusion.34 The present study found that the amplitude of PhNR in mice exposed to an altitude of 5000 m was significantly reduced compared with that of the healthy control group mice. Li et al. found that the loss of RGCs was significantly correlated with the amplitude of PhNR in rats that had undergone optic nerve transection.33 Shen et al. studied the relationship between RNFL thickness and PhNR in healthy patients and patients with primary open-angle glaucoma and found that the amplitude of PhNR was linearly correlated with the structural parameter RNFL thickness.35 Our study demonstrated that the amplitude of PhNR began to significantly decrease from 2 hours of HAE, reaching its lowest value at 6 hours. Meanwhile, there was an increasing trend at 10 and 24 hours compared with 6 hours, followed by a decreasing trend at 72 hours. However, the amplitude of PhNR significantly decreased at various time points after HAE compared with the control group. In addition, the amplitude of PhNR showed a decreasing trend similar to the thickening of the RNF and GCL at different time points after HAE. However, the times when the amplitude of PhNR began to significantly decrease and then reached its lowest value were both earlier than the time when the RNF and GCL began to significantly thicken and reach their peak. Therefore, we believe that RGCs are likely to have already experienced functional decline before undergoing morphological changes during high-altitude hypoxia. Machida performed a prospective study on the relationship between PhNR amplitude and RNF thickness around the optic disc after traumatic optic neuropathy and found that functional loss preceded morphological changes,36 congruous with our conclusion. 
In addition, selecting optimal stimulation conditions for generating PhNR and accurately measuring the amplitude of PhNR is crucial for evaluating the function of RGCs. Recently, the International Society for Clinical Electrophysiology and Vision (ISCEV) recommended in the extended protocol for PhNR that red stimuli on a blue background should be used to record PhNR.37 Compared with broadband stimuli (white stimuli on a white background), monochromatic stimuli (red flashes on a blue background) have been shown to enhance the amplitude of PhNR.35 However, Gotoh et al.38 argued that even traditional W/W stimuli can trigger significant PhNR amplitude as long as it is recorded from a photosensitive system. Rufiange et al.39 believed that the efficiency of the OFF-retinal pathway stimulated by red flashes is lower, resulting in lower b-waves compared with white stimuli. Moreover, depending on the type of electrode and stimulator used, the configuration of stimulation, and the testing environment, each laboratory should set its own PhNR testing parameters according to its conditions. Herein, we used two different stimulation conditions (R/B and W/W) to induce PhNR to evaluate functional changes in RGCs in HAE mice. Our results indicated that the PhNR elicited by W/W was equivalent to that elicited by R/B in evaluating functional damage of RGCs in HAE mice; however, W/W can elicit larger PhNR amplitudes than R/B. Our findings regarding PhNR measurements deviate from the recommendations set forth by the ISCEV extended protocol.37 This difference might be attributed to the absence of red-light sensitive cone cells in the murine retina, which is a physiological distinction between mice and humans. Therefore, we believe that the PhNR recorded under W/W conditions is more suitable for monitoring the functionality of mice RGCs. 
Limitations of This Study
This study has two major limitations. First, rodents exhibit significantly lower tolerance to hypoxia compared with humans, which is related to their hemoglobin oxygen affinity.40 This difference limits our ability to apply research results to humans. Mice represent a valuable preclinical model for studying human diseases due to their high degree of genetic homology, physiological similarity, and well-conserved biological mechanisms. In addition, their economic viability and ease of acquisition make them a practical choice for research. Given the similarities between the retinal vasculature in mice and humans, researchers often utilize mouse models exposed to low-pressure and hypoxic chambers to investigate high-altitude cerebral edema (HACE) and other high-altitude illnesses. Therefore, our study is also valuable. Further exploration of the setting conditions of low-pressure and low oxygen chambers is needed to make the retinal lesions in mice more similar to the clinical manifestations of human high-altitude retinopathy, or to search for animal models that are closer to human hypoxia tolerance, have similar retinal structures to humans, and are easy to obtain. Factors influencing changes in PhNR amplitude extend beyond the retinal ganglion cells themselves. These changes can also be attributed to variations in the signals generated by photoreceptors and bipolar cells. It is difficult to determine whether the origin of PhNR amplitude changes is in the RGCs themselves or at another location in the retina (photoreceptor cells or bipolar cells) when waves a and b undergo changes. Our research faces such research limitations. High-altitude exposure resulted in a decrease in the amplitudes of the a-wave, b-wave, and PhNR in our study. Therefore, the PhNR results do not necessarily tell us anything about the ganglion cells in this particular case. However, histological analysis of retinal tissue sections using H&E staining and TEM following HAE revealed thickening of the GCL and RNFL, along with RGC swelling and ultrastructural changes. Based on the above reasons, we believe that the amplitude of PhNR in mice decreases after HAE, probably due to RGCs damage. 
In summary, damage to RGCs contributes to retinal neuropathy and can directly disrupt the transmission of visual signals along the visual pathway, resulting in impaired vision. In this study, we found that the morphology and function of RGCs were damaged following exposure to high altitude. Therefore, we propose that protecting RGCs against injury could be a promising avenue for treating acute high-altitude retinopathy. 
Acknowledgments
Supported by grants from the National Natural Science Foundation of China (grant numbers 82060180); Gansu Province Clinical Research Center for Ophthalmology (20JR10FA669). 
Disclosure: Y. Yang, None; C. Han, None; Y. Sun, None; X. Zhao, None; Z. Chen, None; L. Zhao, None; Y. Li, None; W. Zhang, None 
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Figure 1.
 
The thickness of the GCL and RNFL was increased with acute high-altitude exposed time prolonged. (A) Representative H&E-staining images of mouse retina (scale = 100 µm). Briefly, 20 points were selected equidistant from the posterior pole of the retina toward the peripheral region on the micrograph of the retina slice magnified at 200 times (the starting point is the junction of the optic nerve and retinal pigment epithelium, and the distance between the points is 25 µm). The thickness of the nerve fiber layer (RNFL) and retinal ganglion cell layer (GCL) was assessed at 20 locations on both the left and right sides of the optic disc on retinal slices. The measurements from each eye were averaged to obtain the final values. To collect representative data, five mice were recorded in each group. (B and C) Representative regions of H&E-stained retinal cross sections of the control and acute high-altitude exposed for 2, 4, 6, 10, 24, and 72 hours. Images were captured at the same magnification. Scale bar = 100 µm. (D) Calculation of thickness of the GCL and RNFL in the control group and high-altitude exposed groups at 2, 4, 6, 10, 24, and 72 hours. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group.
Figure 1.
 
The thickness of the GCL and RNFL was increased with acute high-altitude exposed time prolonged. (A) Representative H&E-staining images of mouse retina (scale = 100 µm). Briefly, 20 points were selected equidistant from the posterior pole of the retina toward the peripheral region on the micrograph of the retina slice magnified at 200 times (the starting point is the junction of the optic nerve and retinal pigment epithelium, and the distance between the points is 25 µm). The thickness of the nerve fiber layer (RNFL) and retinal ganglion cell layer (GCL) was assessed at 20 locations on both the left and right sides of the optic disc on retinal slices. The measurements from each eye were averaged to obtain the final values. To collect representative data, five mice were recorded in each group. (B and C) Representative regions of H&E-stained retinal cross sections of the control and acute high-altitude exposed for 2, 4, 6, 10, 24, and 72 hours. Images were captured at the same magnification. Scale bar = 100 µm. (D) Calculation of thickness of the GCL and RNFL in the control group and high-altitude exposed groups at 2, 4, 6, 10, 24, and 72 hours. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with the control group.
Figure 2.
 
The loss of RGCs in both the central and peripheral regions of the retina increases with prolonged exposure to high altitude. (A) Whole-mount retinas were immunostained with Brn3a and RGCs were quantified in two sampling areas (at 500 µm from the optic nerve head for the center and 500 µm from the peripheral edge for the periphery) in each of the four quadrants of the retina. Scale bar = 500 µm. (B) Representative high-magnification images of BRN3A immunostaining RGCs from the central or peripheral retina at the control group and high-altitude exposure for 2, 4, 6, 10, 24, and 72 hours. Scale bar = 100 µm. (C) Quantitative analysis of BRN3A+ immunostaining RGCs density in the retinal center. (D) Quantitative analysis of BRN3A+ immunostaining RGCs density in the retinal periphery. Data are the mean ± SEM; * P < 0.05 versus the control.
Figure 2.
 
The loss of RGCs in both the central and peripheral regions of the retina increases with prolonged exposure to high altitude. (A) Whole-mount retinas were immunostained with Brn3a and RGCs were quantified in two sampling areas (at 500 µm from the optic nerve head for the center and 500 µm from the peripheral edge for the periphery) in each of the four quadrants of the retina. Scale bar = 500 µm. (B) Representative high-magnification images of BRN3A immunostaining RGCs from the central or peripheral retina at the control group and high-altitude exposure for 2, 4, 6, 10, 24, and 72 hours. Scale bar = 100 µm. (C) Quantitative analysis of BRN3A+ immunostaining RGCs density in the retinal center. (D) Quantitative analysis of BRN3A+ immunostaining RGCs density in the retinal periphery. Data are the mean ± SEM; * P < 0.05 versus the control.
Figure 3.
 
The ultrastructural images of retinal RGCs from C57BL/6 mice. Normal ultrastructure of RGCs with intact cell structure, smooth nuclear membrane, uniform distributed chromatin, moderate electron density in the cytoplasm, and clear mitochondrial structure in the control group. RGCs exhibited abnormal cellular structure, including nuclear membrane abnormalities, uneven distribution of chromatin in the nucleus, decreased cytoplasmic electron density, disordered mitochondrial structure with increased volume, widened membrane gaps, and reduced cristae at 2 to72 hours after high-altitude exposure. Au = autophagosome; M = mitochondria. Scale bar = 2 µm (upper panel) and 1 µm (lower panel).
Figure 3.
 
The ultrastructural images of retinal RGCs from C57BL/6 mice. Normal ultrastructure of RGCs with intact cell structure, smooth nuclear membrane, uniform distributed chromatin, moderate electron density in the cytoplasm, and clear mitochondrial structure in the control group. RGCs exhibited abnormal cellular structure, including nuclear membrane abnormalities, uneven distribution of chromatin in the nucleus, decreased cytoplasmic electron density, disordered mitochondrial structure with increased volume, widened membrane gaps, and reduced cristae at 2 to72 hours after high-altitude exposure. Au = autophagosome; M = mitochondria. Scale bar = 2 µm (upper panel) and 1 µm (lower panel).
Figure 4.
 
Ultrastructural images of axons in RGCs from C57BL/6 mice. Normal ultrastructure of axons in RGCs of the control group, with clear hierarchy, dense and regular arrangement of myelin sheaths, tightly connected myelin sheath and axial membrane, well arranged microtubules and nerve fibers, and normal structure mitochondria. The ultrastructure of axons within RGCs displayed abnormalities between 2 and 72 hours following high-altitude exposure. These abnormalities included widened and vacuolized gaps between axons, loosening and disorganization of the myelin sheath structure, widening of the gap between the myelin sheath and axon membrane, reduced axoplasmic density, indistinct microtubule and nerve fiber structure, and irregular mitochondrial morphology (mostly swollen, with reduced cristae and vacuolization). Au = autophagosome; M = mitochondria. Scale bar = 1 µm (upper panel) and 500 nm (lower panel).
Figure 4.
 
Ultrastructural images of axons in RGCs from C57BL/6 mice. Normal ultrastructure of axons in RGCs of the control group, with clear hierarchy, dense and regular arrangement of myelin sheaths, tightly connected myelin sheath and axial membrane, well arranged microtubules and nerve fibers, and normal structure mitochondria. The ultrastructure of axons within RGCs displayed abnormalities between 2 and 72 hours following high-altitude exposure. These abnormalities included widened and vacuolized gaps between axons, loosening and disorganization of the myelin sheath structure, widening of the gap between the myelin sheath and axon membrane, reduced axoplasmic density, indistinct microtubule and nerve fiber structure, and irregular mitochondrial morphology (mostly swollen, with reduced cristae and vacuolization). Au = autophagosome; M = mitochondria. Scale bar = 1 µm (upper panel) and 500 nm (lower panel).
Figure 5.
 
(A and B) Representative PhNR elicited by the W/W and R/B stimuli in the control group and high-altitude exposure group with different exposure times. (C) The amplitudes of a-wave, b-wave, and PhNR elicited by W/W stimuli in the RGCs at control and at each time point after high-altitude exposure (2 hours, 4 hours, 6 hours, 10 hours, 24 hours, and 72 hours) are compared. When compared to the control group, the reduction of PhNR amplitude in each high-altitude exposure time point was statistically significant. Data are presented as the mean ± SEM; ***P < 0.001 and ****P < 0.0001 versus the control. (D) The amplitudes of a-wave, b-wave, and PhNR elicited by R/B stimuli in the RGCs at control and at each time point after high-altitude exposure (2 hours, 4 hours, 6 hours, 10 hours, 24 hours, and 72 hours) are compared. The amplitude of PhNR significantly decreased compared to the control group at various time points after high-altitude exposure except for 2 hours. Data are presented as the mean ± SEM; **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus the control group.
Figure 5.
 
(A and B) Representative PhNR elicited by the W/W and R/B stimuli in the control group and high-altitude exposure group with different exposure times. (C) The amplitudes of a-wave, b-wave, and PhNR elicited by W/W stimuli in the RGCs at control and at each time point after high-altitude exposure (2 hours, 4 hours, 6 hours, 10 hours, 24 hours, and 72 hours) are compared. When compared to the control group, the reduction of PhNR amplitude in each high-altitude exposure time point was statistically significant. Data are presented as the mean ± SEM; ***P < 0.001 and ****P < 0.0001 versus the control. (D) The amplitudes of a-wave, b-wave, and PhNR elicited by R/B stimuli in the RGCs at control and at each time point after high-altitude exposure (2 hours, 4 hours, 6 hours, 10 hours, 24 hours, and 72 hours) are compared. The amplitude of PhNR significantly decreased compared to the control group at various time points after high-altitude exposure except for 2 hours. Data are presented as the mean ± SEM; **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus the control group.
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