Long-Term Functional and Structural Consequences of Primary Blast Overpressure to the Eye

Abstract

Acoustic blast overpressure (ABO) injury in military personnel and civilians is often accompanied by delayed visual deficits. However, most animal model studies dealing with blast-induced visual defects have focused on short-term (≤1 month) changes. Here, we evaluated long-term (≤8 months) retinal structure and function deficits in rats with ABO injury. Adult male Long-Evans rats were subjected to ABO from a single blast (approximately 190 dB SPL, ∼63 kPa, @80 psi), generated by a shock tube device. Retinal function (electroretinography; ERG), visual function (optomotor response), retinal thickness (spectral domain-optical coherence tomography; SD-OCT), and spatial cognition/exploratory motor behavior (Y-maze) were measured at 2, 4, 6, and 8 months post-blast. Immunohistochemical analysis of glial fibrillary acidic protein (GFAP) in retinal sections was performed at 8 months post-blast. Electroretinogram a- and b-waves, oscillatory potentials, and flicker responses showed greater amplitudes with delayed implicit times in both eyes of blast-exposed animals, relative to controls. Contrast sensitivity (CS) was reduced in both eyes of blast-exposed animals, whereas spatial frequency (SF) was decreased only in ipsilateral eyes, relative to controls. Total retinal thickness was greater in both eyes of blast-exposed animals, relative to controls, due to increased thickness of several retinal layers. Age, but not blast exposure, altered Y-maze outcomes. GFAP was greatly increased in blast-exposed retinas. ABO exposure resulted in visual and retinal changes that persisted up to 8 months post-blast, mimicking some of the visual deficits observed in human blast-exposed patients, thereby making this a useful model to study mechanisms of injury and potential treatments.

Keywords: blast; electroretinogram; optomotor response; retina; visual function.

FIG. 1.

Acoustic blast overpressure setup. (A,B) Shock tube assembly. (C) Compressed gas tank and controller unit, outside blast chamber. (D-F) Placement of rat in holder and positioning prior to blast. (G) Brass foil diaphragm before (lower image) and after (upper image) blast. (H) Pressure sensor assembly, end of shock tube, and animal holder.

FIG. 2.

Representative ERG waveforms from control rats (blue traces) versus ipsilateral (ipsi) and contralateral (contra) eyes from blast-exposed rats (red traces) at 8 months post-blast. Blast-exposed rats exhibit increased amplitudes and delayed implicit times in both ipsilateral and contralateral eyes, compared with age-matched controls. (A) Dark-adapted ERGs; (B) dark-adapted oscillatory potentials; (C) light-adapted flicker responses. ERG, electroretinography.

FIG. 3.

Quantification of ERG a-wave, b-wave, oscillatory potentials, and flicker response amplitudes and implicit times. Significant increases in dark-adapted a-wave (A), dark-adapted b-wave (C), dark-adapted oscillatory potential (E), and light-adapted flicker (G) amplitudes were observed in both ipsilateral (OD) and contralateral (OS) eyes of blast-exposed rats compared with non-blast-exposed controls. Significant delays in dark-adapted a-wave (B), dark-adapted b-wave (D), dark-adapted oscillatory potential (F), and light-adapted flicker implicit times (H) were observed in both eyes from blast-exposed rats compared with controls; ***p < 0.001, **p < 0.01. Results expressed as mean ± SEM values. ERG, electroretinography; SEM, standard error of the mean.

FIG. 4.

Reduced spatial frequency and contrast sensitivity in blast-exposed versus control rats. (A) Spatial frequency thresholds as a function of post-blast time (2–8 months). (B) Contrast sensitivity thresholds as a function of post-blast time (2–8months). Black asterisks indicate comparisons of ipsilateral blast-exposed eyes (OD) with both the contralateral (OS) blast-exposed eyes and both eyes of control rats; blue asterisks indicate comparisons of the contralateral (OS) blast-exposed eyes with both the ipsilateral (OD) blast-exposed eyes and with both eyes of control rats. ***p < 0.001. Results expressed as mean ± SEM values. SEM, standard error of the mean.

FIG. 5.

Representative SD-OCT images taken 4 months post-blast from (A) control and (B) blast-exposed rats. Lines delineating retinal nerve fiber layer (RNFL) to easily visualize increased layer thickness of blast-exposed eye versus control. Total retinal thickness (TRT) was measured from the top of the RNFL to the bottom of the RPE. ELM, external limiting membrane; INL, inner nuclear layer; IPL, inner plexiform layer; IS/OS, inner segment/outer segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium; SD-OCT, spectral domain–optical coherence tomography.

FIG. 6.

Quantification of SD-OCT measurements by quadrant and across time. Significant increase in total retinal thickness (A) of blast versus control animals (eyes averaged per group) due to increases in individual layers of the retina, including: RNFL (B), OPL (C), ONL (D), INL (E) and IS/OS (F). IS/OS showed significant changes in the contralateral eye of blast-exposed animals as well. Blue asterisks indicate comparisons of ipsilateral blast-exposed eyes (OD) and contralateral (OS) blast-exposed eyes with both eyes of control rats; black asterisks indicate comparisons of contralateral (OS) blast-exposed eyes with both the ipsilateral blast-exposed eyes (OD) and with both eyes of control rats. ***p < 0.001. Results expressed as mean ± SEM values. INL, inner nuclear layer; IS/OS, inner segment/outer segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; SD-OCT, spectral domain–optical coherence tomography; SEM, standard error of the mean.

FIG. 7.

Marked upregulation and persistence of anti-GFAP immunoreactivity in ipsilateral blast-exposed retina (C,D), relative to non-blast-exposed control (A,B). Frozen tissue sections were immunostained with anti-GFAP, followed by treatment with AlexFluor^® 647-conjugated secondary antibody (magenta), and counterstained with DAPI (blue) to label nuclei. Laser confocal fluorescence microscopy images were taken, with (A,C) and without (B,D) the DAPI channel. A representative area of peri-central retina for each condition is shown. Scale bar (panel D), 25 μm. ILM, inner limiting membrane; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; GFAP, glial fibrillary acidic protein; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium.

FIG. 8.

Y-maze analysis of (A) cognitive function and (B) exploratory motor behavior of blast-exposed (red trace) versus control rats (blue trace) as a function of post-blast time. Both groups exhibited comparable performance measures, and both groups also exhibited performance decline (deficits) over time. Assessments were made at 3, 6, and 8 months post-blast. Cognitive function was measured by spatial alternation on Y-maze; exploratory motor behavior was measured by number of entries on Y-maze. Results expressed as mean ± SEM values. SEM, standard error of the mean.