Traumatic Optic Neuropathy Is Associated with Visual Impairment, Neurodegeneration, and Endoplasmic Reticulum Stress in Adolescent Mice

Abstract

Traumatic brain injury (TBI) results in a number of impairments, often including visual symptoms. In some cases, visual impairments after head trauma are mediated by traumatic injury to the optic nerve, termed traumatic optic neuropathy (TON), which has few effective options for treatment. Using a murine closed-head weight-drop model of head trauma, we previously reported in adult mice that there is relatively selective injury to the optic tract and thalamic/brainstem projections of the visual system. In the current study, we performed blunt head trauma on adolescent C57BL/6 mice and investigated visual impairment in the primary visual system, now including the retina and using behavioral and histologic methods at new time points. After injury, mice displayed evidence of decreased optomotor responses illustrated by decreased optokinetic nystagmus. There did not appear to be a significant change in circadian locomotor behavior patterns, although there was an overall decrease in locomotor behavior in mice with head injury. There was evidence of axonal degeneration of optic nerve fibers with associated retinal ganglion cell death. There was also evidence of astrogliosis and microgliosis in major central targets of optic nerve projections. Further, there was elevated expression of endoplasmic reticulum (ER) stress markers in retinas of injured mice. Visual impairment, histologic markers of gliosis and neurodegeneration, and elevated ER stress marker expression persisted for at least 30 days after injury. The current results extend our previous findings in adult mice into adolescent mice, provide direct evidence of retinal ganglion cell injury after head trauma and suggest that axonal degeneration is associated with elevated ER stress in this model of TON.

Keywords: ER stress; adolescent head trauma; head trauma; mice; traumatic optic neuropathy.

Figure 1

Experimental outline. Two cohorts of mice were utilized in this study. (A) shows a timeline for described experiments. (B) shows an estimate of our weight drop location with scalp intact. Arrows indicate the location of bregma. OMM = Optomotor Machine. Images in (B) were created in BioRender at BioRender.com.

Figure 2

Weight, righting, and morbidity. For cohort 1 (A) and cohort 2 (B), there were no differences between sham and TBI in weight at any time up to 8 days after injury. Survival was not affected by the starting weight for cohort 1 (C) or cohort 2 (D). (E) The two mice who experienced seizures had significantly longer righting times (β = 0.79). * indicates p < 0.05.

Figure 3

TBI/iTON mice have significantly blunted optokinetic responses. The number of responses were totaled for both left and right directions are represented as mean ± SEM for each spatial frequency analyzed. (A) Photograph of the behavioral testing apparatus. (B) Shows an example of a visual grating as used in this test. (C) Optokinetic responses at 7 DPI. (D) Optokinetic responses at 30 DPI. * p < 0.001 and # p < 0.005 vs. sham at the same grating size.

Figure 4

There are no circadian rhythm deficits or histological changes in the suprachiasmatic nucleus of iTON mice. Show is Fluoro-jade B (A,B), GFAP expression (C,D), and microglial soma area (E,F) in the Suprachiasmatic Nucleus (SCN). (G) Shows activity monitoring results. Representative photomicrographs taken at 10× magnification. White dashed lines indicate region of interest/area where measurements were taken. Scale bars indicate 100 µm and are the same across all images.

Figure 5

Retinal ganglion cell immunohistochemistry. Retinas of sham and TBI mice were immunolabeled with Brn3a (red), GFAP (green), and DAPI (blue). Brn3a-labeled cells were counted in 3 zones of the retina (A,D,G) Representative photomicrographs taken at 20× magnification of sham (B,E,H) and TBI/iTON mice (C,F,I). (J) Depiction of whole-mounted retina with grey squares representing photo locations. Retinas were divided into three regions: peripheral (P), mid-peripheral (MP), and central (C). The red circle indicates the location of the optic nerve. Scale bar indicates 100 μm and is the same for all images, * p < 0.001.

Figure 6

Retinal ganglion cell counts at 30 DPI. Retinas of sham and TBI mice were labeled with Brn3a (red), GFAP (green), and DAPI (blue). Cell count results are shown for center (A), mid-peripheral (D), and peripheral (G). Representative photomicrographs taken at 20× magnification of sham (B,E,H) and TBI/iTON mice (C,F,I). Scale bar indicates 100 μm and is the same for all images, * p < 0.001.

Figure 7

Neurodegeneration in the optic system 7 (A–D) and 30 (E–H) DPI. Staining appears punctate rather than somatic, consistent with primarily axonal staining. Representative photomicrographs taken at 10× magnification with a scale bar indicating 100 μm; this applies to all images. * p < 0.001 and # p <0.05.

Figure 8

Morphologic evidence of microglial activation in the optic system is present 7 (A–D) and 30 (E–H) DPI. These morphologic changes are particularly predominant in the OT but can also clearly be seen in other regions (red arrows). Representative photomicrographs taken at 10× magnification, scale bar 100 μm. # p < 0.05. Higher magnification images can be found in Supplementary Figure S3, * p < 0.001.

Figure 9

Astrogliosis of the optic system in iTON mice 7 (A–D) and 30 (E–H) DPI. White dashed lines mark the areas analyzed in sham and iTON mice where it is more difficult to visualize the location of the nuclei. Representative photomicrographs taken at 10× magnification, scale bar 100 μm. * p ≤ 0.001 and # p < 0.05.

Figure 10

Markers of apoptosis and endoplasmic reticulum stress in the retinas of TBI/iTON mice 7 DPI. (A) Shows markes implicated in ER stress, (B) shows elevated Caspase-3 (an apoptotic marker). Protein measurements were normalized to beta-actin. * p < 0.05.

Figure 11

Markers of endoplasmic reticulum stress (A) and caspase-3 expression (B) at 30 DPI. * p < 0.05.