Elevated intraocular pressure causes inner retinal dysfunction before cell loss in a mouse model of experimental glaucoma

Abstract

Purpose: We assessed the relationship among intraocular pressure (IOP), histology, and retinal function changes in a mouse model of induced, chronic, mild ocular hypertension.

Methods: IOP was elevated experimentally via anterior chamber injection of polystyrene beads and measured twice weekly with a rebound tonometer. Histology was assessed with a combination of neurobiotin (NB) retrograde labeling of retinal ganglion cells (RGCs) and TO-PRO3 staining. Retinal function was assessed with serial dark-adapted electroretinograms (ERGs) optimized for detection of the a-wave, b-wave, and positive and negative scotopic threshold responses (pSTR, nSTR). Comparisons between bead-injected and saline-injected (control) eyes were conducted.

Results: IOP remained elevated for at least 3 months following a single injection of polystyrene beads. Elevated IOP resulted in a mild, progressive reduction of RGCs, and a mild increase in axial length at 6 and 12 weeks after bead injection. The raw b-wave amplitude was increased shortly after IOP elevation, but the raw a-wave, pSTR, and nSTR amplitudes were unchanged. pSTR and nSTR amplitudes were normalized to the increased b-wave. With this normalization, the pSTR amplitude was decreased shortly after IOP elevation.

Conclusions: Polystyrene bead injection results in a mild, chronic elevation of IOP that recapitulates several critical aspects of human ocular hypertension and glaucoma, and results in early changes in retinal electrical function that precede histologic changes. It is possible that glaucoma associated with elevated IOP involves the early disruption of a complex combination of retinal synapses.

Conflict of interest statement

Disclosure: B.J. Frankfort, None; A.K. Khan, None; D.Y. Tse, None; I. Chung, None; J.-J. Pang, None; Z. Yang, None; R.L. Gross, None; S.M. Wu, None

Figures

Figure 1.

Patterns of IOP. IOP is elevated in bead-injected eyes. (A) Mean IOP for bead-injected (green) and uninjected (blue) eyes as measured twice weekly over 12 weeks (N = 96 animals through postoperative day [POD] 43, N = 33 from PODs 47–85, see Table 1). IOP is elevated by POD 1 and remains elevated at every measured time point for at least 12 weeks (t-test, P < 0.05). The difference in IOP between bead-injected and uninjected eyes is presented in red. (B) Mean cumulative IOP difference between bead-injected and saline-injected eyes. The cumulative IOP difference for each animal was determined according to Equation 1 (see Materials and Methods) and used to calculate the mean cumulative IOP difference (mm Hg). Bead-injected eyes are subject to a larger final cumulative IOP than saline-injected eyes at all measured time points (t-test, P < 0.01). Error bars represent one SEM for panels.

Figure 2.

Inner retina staining. Neurobiotin-positive (red) and TO-PRO3–positive (green) cells were counted from 8 regions per retina (middle, red squares). Left: typical staining pattern from the midretina. Right: typical staining pattern from the peripheral retina.

Figure 3.

ERG analysis. IOP elevation results in b-wave abnormalities. (A–G). 20 bead-injected and 5 saline-injected animals were followed with sequential bilateral simultaneous ERGs (see Materials and Methods) at 2-week intervals beginning at 2 weeks postinjection and continuing for 12 weeks (postoperative weeks [POWs] 2–12). (A–D). Representative tracings at increasing light stimuli (log intensity scale, see Materials and Methods). For all tracings, the x-axis represents msec and the y-axis represents μV. Note the change in scale of the y-axis for each tracing to highlight best the individual waveforms. (A) At low scotopic range light intensities the pSTR and nSTR are observed. (B) At slightly greater light intensities a transition wave is detected. (C) At slightly greater light intensities the b-wave is first observed, and the pSTR and nSTR are no longer detected. (D) At brighter light intensities the a-wave and b-wave are detected. (E) Raw amplitudes of ERG waveforms obtained from bead-injected, saline-injected, and uninjected eyes. For the a-wave and b-waves, amplitudes obtained at six stimulus levels were compared for all stimuli and at all time points, and then averaged for presentation. For the pSTR and the nSTR, amplitudes obtained at two stimulus levels were compared for stimuli and at all time points, and then averaged for presentation (see text, Materials and Methods). The b-wave amplitude of bead-injected eyes is increased compared to either saline-injected or uninjected eyes, whereas the a-wave, pSTR, and nSTR amplitudes are unchanged (ANOVA P < 0.001). (F) The b-wave amplitude difference was calculated by subtracting the b-wave amplitude of the uninjected eye from the b-wave amplitude of the injected eye (beads or saline) for each animal at every time point. This difference is positive, indicating an increased b-wave amplitude in bead-injected eyes at all time points. (G) The b-wave amplitude ratio was calculated according to Equation 2 (see Materials and Methods). Bead-injected eyes have an increased mean b-wave amplitude ratio compared to saline-injected eyes at all time points. (F, G) suggest that the intra-animal b-wave amplitude is increased rapidly for bead-injected eyes at each time point (t-test, * = P < 0.05). Error bars represent one SEM for all panels.

Figure 4.

STR analysis. Normalized STR amplitudes are reduced when IOP is elevated. (A–C). The normalized pSTR and nSTR difference for each animal was calculated according to Equation 4 (see Materials and Methods). (A) Bead-injected eyes have a reduced mean normalized pSTR difference when compared to saline-injected eyes beginning at POW 4 (ANOVA P < 0.001, t-test, * = P < 0.05). (B) The bead-injected animals were split into two equal groups according to the median cumulative IOP difference (see Results). The group with the higher cumulative IOP (Beads, High IOP) had a reduced mean normalized pSTR difference beginning at POW 4, while the group with the lower cumulative IOP (Beads, Low IOP) had a reduced mean normalized pSTR difference beginning at POW6 (t-test, * = P < 0.05), suggesting that there is an IOP-dependent effect on normalized pSTR change. (C) Bead-injected eyes have a reduced mean normalized nSTR difference (ANOVA P < 0.05). However, confirmatory t-tests performed at each time point were not statistically significant, suggesting a more mild IOP-dependent effect on the normalized nSTR than for the normalized pSTR. Error bars represent one SEM for all panels.