Longitudinal Photoreceptor Phenotype Observation and Therapeutic Evaluation of a Carbonic Anhydrase Inhibitor in a X-Linked Retinoschisis Mouse Model

Abstract

Purpose: To study the long-term photoreceptor changes and to evaluate the effects of topical application of a carbonic anhydrase inhibitor (CAI) in a mouse model of X-linked retinoschisis (XLRS).

Methods: Conventional electroretinograms (ERGs) and dark-adapted 10-Hz flicker ERGs were recorded in control and Rs1 ^-/Y mice generated with CRISPR/Cas9. ON-pathway blocker 2-amino-4-phosphobutyric acid (APB) was injected intravitreally. Morphology was evaluated with histology and optical coherence tomography (OCT). Mice were treated with a CAI inhibitor brinzolamide eye drops (10 mg/ml) three times a day for 3 months. OCT and ERG findings at 1, 4, and 10 months were analyzed.

Results: Negative ERGs and retinal cavities were evident in Rs1 ^-/Y mice. Both a-wave and b-wave amplitudes decreased with age when compared with age-matched controls. The APB-isolated a-wave (a') amplitudes of Rs1 ^-/Y mice were reduced in all age groups. In dark-adapted 10-Hz flicker ERG, the amplitude-intensity curve of Rs1 ^-/Y mice shifted down. The thickness of ONL and IS/OS decreased in Rs1 ^-/Y mice. CAI reduced the splitting retinal cavities but didn't affect the ERG.

Conclusions: In addition to post receptoral impairments, photoreceptor cells underwent progressive dysfunction since early age in Rs1 ^-/Y mice. Long-term CAI treatment improved the shrinkage of the splitting retinal cavity, while no functional improvement was observed.

Keywords: ERG; Rs1; X-linked juvenile retinoschisis; carbonic anhydrase inhibitor; mouse; retinal degeneration.

Figure 1

Rs1^−/Y mouse model generation and genotyping strategy. CRISPR/Cas9 genome editing system with two guide RNAs was used to generate the Rs1^−/Y mouse model. (A) In the targeting construct, all coding sequences of Rs1 gene were deleted. Forward primer of genotyping located at the upsteam of gRNA-1 and reverse primer located at the downstream of gRNA-2. (B) gRNA sequencing of Rs1 knockout mice. (C) DNA sequencing analysis revealed 28 kb deletion in one founder. (D) The absence of normal Rs1 protein in retinal extracts of Rs1^−/Y mice.

Figure 2

PCR amplification demonstrates the correct targeting of Rs1gene. One band of 350 bp was shown in WT, one band of 670 bp was shown in Rs1^−/Y mice, and both bands of 350 and 670 bp were shown in heterozygote.

Figure 3

Representative ERG amplitude changes with age in Rs1^−/Y mice and age-matched WT mice. (A) A series of dark-adapted ERGs of Rs1^−/Y and WT mice of different ages (1, 4, 6, and 10 months). (B) a-Wave V-log I, b-wave V-log I. n = 9 at each age group. WT, wild type; ERG, electroretinogram.

Figure 4

The effect of APB on the dark-adapted ERG of Rs1^−/Y mice and and age-matched WT mice. (A) The black traces show ERGs of the PBS-injected control eyes and the red traces show ERGs of the APB injected eyes. In the APB-injected eye, the a-wave remains but the b-waves are absent. (B) Left: The a′ wave amplitude changes with age in Rs1^−/Y mice of 1, 6, and 10 month old. ERG stimulus, 0 log cd-s/m². (C) Right: a′/a ratio in Rs1^−/Y mice of three different ages (1, 6, and 10 months). Dashed lines: the a′/a ratio mean ± SE of WT: 1.190 ± 0.127 n = 5. a′ wave: the negative wave in APB injected eye; APB, 2-amino-4-phosphobutyric acid. Rs1^−/Y results compared to WT using Student's t-test: ^***, P < 0.001.

Figure 5

Dark-adapted 10-Hz flicker ERGs elicited with a series of light intensity in WT mice of different ages.

Figure 6

Dark-adapted 10-Hz flicker ERGs elicited with a series of light intensity in Rs1^−/Y mice of different ages.

Figure 7

Dark-adapted 10-Hz flicker ERG response amplitude-intensity profiles (n = 3 at each age group).

Figure 8

Morphology changes in Rs1^−/Y mouse and age-matched WT mice. (A) Representative sections from retinas of 1, 6, and 10-month-old Rs1^−/Y mice and age-matched WT mice and retinal images obtained from OCT were used to evaluate the retinal morphology at three different ages. (B) The thickness of the ONL, INL, OS+IS 300 μm away from the optic nerve was measured (n = 4 at each age group). (C) a-Wave amplitude vs. OS+IS thickness. Lines: linear regression (R² = 0.7590) and 95% confidence intervals. a-Wave amplitude vs. ONL thickness. Lines: linear regression (R² = 0.6868) and 95% confidence intervals, P < 0.0001; ONL, outer nuclear layer; INL, inner nuclear layer; OS, outer segment; IS, inner segment. Rs1^−/Y results compared to WT using Student's t-test: ^*, P < 0.05; ^**, P < 0.01; ^***, P < 0.001.

Figure 9

Structural and functional changes after CAI treatment in Rs1^−/Y mice retina were evaluated by OCT and ERG. (A) Retinal images obtained from OCT in living animals were used to evaluate the retinal morphology at 3 different ages. From 1 month, the left eyes of Rs1^−/Y mice were treated with brinzolamide eye drops (10 mg/ml) for 3 continuous months, the contralateral eyes were treated with PBS as control. OCT were performed at 1, 4, 10 months of age. (B) ONL and INL thickness were measured from 200 to 1,200 μm inferior and superior to the optic nerve (n = 6 at each time point). (C) Functional changes by CAI treatment in Rs1^−/Y mice retina were evaluated by ERG. The CAI treated eyes were compared with the control eyes using Student's t-test: ^***, P < 0.001. CAI, carbonic anhydrase inhibitor; OCT, optical coherence tomography.