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. 2017 Feb 10:11:41.
doi: 10.3389/fnins.2017.00041. eCollection 2017.

Characterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels

Da Zhao  1 , Christine T O Nguyen  1 , Vickie H Y Wong  1 , Jeremiah K H Lim  1 , Zheng He  1 , Andrew I Jobling  2 , Erica L Fletcher  2 , Holly R Chinnery  1 , Algis J Vingrys  1 , Bang V Bui  1
Affiliations

Characterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels

Da Zhao et al. Front Neurosci. .

Abstract

To consider whether a circumlimbal suture can be used to chronically elevate intraocular pressure (IOP) in mice and to assess its effect on retinal structure, function and gene expression of stretch sensitive channels. Anesthetized adult C57BL6/J mice had a circumlimbal suture (10/0) applied around the equator of one eye. In treated eyes (n = 23) the suture was left in place for 12 weeks whilst in sham control eyes the suture was removed at day two (n = 17). Contralateral eyes served as untreated controls. IOP was measured after surgery and once a week thereafter. After 12 weeks, electroretinography (ERG) was performed to assess photoreceptor, bipolar cell and retinal ganglion cell (RGC) function. Retinal structure was evaluated using optical coherence tomography. Retinae were processed for counts of ganglion cell density or for quantitative RT-PCR to quantify purinergic (P2x7, Adora3, Entpd1) or stretch sensitive channel (Panx1, Trpv4) gene expression. Immediately after suture application, IOP spiked to 33 ± 3 mmHg. After 1 day, IOP had recovered to 27 ± 3 mmHg. Between weeks 2 and 12, IOP remained elevated above baseline (control 14 ± 1 mmHg, ocular hypertensive 19 ± 1 mmHg). Suture removal at day 2 (Sham) restored IOP to baseline levels, where it remained through to week 12. ERG analysis showed that 12 weeks of IOP elevation reduced photoreceptor (-15 ± 4%), bipolar cell (-15 ± 4%) and ganglion cell responses (-19 ± 6%) compared to sham controls and respective contralateral eyes (untreated). The retinal nerve fiber layer was thinned in the presence of normal total retinal thickness. Ganglion cell density was reduced across all quadrants (superior -12 ± 5%; temporal, -7% ± 2%; inferior -9 ± 4%; nasal -8 ± 5%). Quantitative RT-PCR revealed a significant increase in Entpd1 gene expression (+11 ± 4%), whilst other genes were not significantly altered (P2x7, Adora3, Trpv4, Panx1). Our results show that circumlimbal ligation produces mild chronic ocular hypertension and retinal dysfunction in mice. Consistent with a sustained change to purinergic signaling we found an up-regulation of Entpd1.

Keywords: P2X7; TRPV; eNTDPase; electroretinography; glaucoma; intraocular pressure; pannexin; retinal ganglion cells.

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Figures

Figure 1
Figure 1
Chronic intraocular pressure (IOP) elevation in mouse eyes. (A) Group IOP (mean ± standard error of the mean) in ocular hypertensive (OHT, red n = 23) and their untreated contralateral control eyes (C-OHT, unfilled). Inset: Image of a mouse eye with a circumlimbal suture (arrowheads) in place. (B) IOP in eyes that had the suture removed on day 2 (Sham, blue n = 17) and their untreated contralateral control eyes (C-Sham, gray).
Figure 2
Figure 2
Effect of circumlimbal suture and chronic intraocular pressure (IOP) elevation on the mouse anterior segment. (A–C) Representative en face images of an ocular hypertensive (B. OHT), its fellow control (A. C-OHT) eye, and a sham operated eye (C. Sham) at week 12, acquired during anterior segment imaging allowing pupil diameter to be quantified. (D–F) Sagittal optical coherence tomography images of the anterior chamber for an OHT (E), its control eye (D), and a sham operated eye (F). (G) Averaged (± SEM) pupil size for OHT (n = 23, red), C-OHT (unfilled) as well as Sham (n = 17, blue), and their contralateral control eyes (C-Sham, gray). (H) Average trabecular meshwork-iris angle (TIA). (I) Average anterior chamber depth.
Figure 3
Figure 3
Effect of chronic circumlimbal suture on retinal blood flow. (A,C) Fundus images of a control and the treated eye in the OHT group. (B,D) Representative images of Doppler blood flow annular scan in the same C-OHT and OHT eyes. (E) Group average (± SEM) total blood flow signal in OHT (n = 23, red) and Sham (n = 17, blue) treated eyes and their contralateral controls (C-OHT unfilled, C-Sham gray). (F) Group average arterial signal. (G) Group average venous signal.
Figure 4
Figure 4
Effect of chronic IOP elevation on ERG waveforms. (A) Ocular hypertensive (OHT, red traces, n = 23) and control eyes (C-OHT, black traces) (B) Sham (blue traces, n = 17) and control eyes (C-Sham, black traces).
Figure 5
Figure 5
Effect of chronic IOP elevation on retinal function. (A) Average (± SEM) photoreceptoral (P3) amplitude for OHT (red bar, n = 23) and Sham (blue, n = 17) as well as their respective contralateral control eyes (C-OHT, unfilled; C-Sham, gray). (B) Photoreceptor P3 sensitivity (S). (C) ON-bipolar cell amplitude (P2). (D) ON-bipolar cell (P2) sensitivity (1/k). (E) Inner retinal inhibitory circuits [Oscillatory Potentials (OPs)]. (F) OP peak implicit time. (G) Ganglion cell mediated positive scotopic threshold response (pSTR). (H) pSTR implicit time. **p < 0.001, *p < 0.05.
Figure 6
Figure 6
Effect of chronic IOP elevation on retinal structure. (A,B) Representative OCT images of a sutured eye and the control eye of a mouse in the OHT group. Bruch's membrane opening width (BMO), minimum rim thickness (MRT), total retinal thickness (TRT), and retinal nerve fibre layer (RNFL) thickness were measured on both temporal and nasal sides of the nerve and averaged. (C) Average (± SEM) RNFL thickness for OHT (red, n = 23) and Sham (blue, n = 17) as well as their respective contralateral control eyes (C-OHT, unfilled; C-Sham, gray). (D) Group average MRT. (E) Group average TRT. (F) Group average BMO. **p < 0.001, *p < 0.05.
Figure 7
Figure 7
Effect of 12 weeks of chronic IOP elevation on RGC density. (A,B) Representative micrographs of RGCs stained with RBPMS in an OHT eye and its contralateral control eye. (C) Average (± SEM) RGC density on the superior retina, for OHT (red, n = 12) and Sham (blue, n = 8) as well as their respective contralateral control eyes (C-OHT, unfilled; C-Sham, gray). (D) RGC density in the temporal retina. (E) RGC density in the inferior retina. (F) RGC density in the nasal retina. **P < 0.001, *P < 0.05.
Figure 8
Figure 8
Effect of 12 weeks of chronic IOP elevation on expression of pressure sensitive cell membrane receptors assayed 12 weeks after IOP elevation. (A) Average (± SEM) P2x7 gene expression relative to Hprt for OHT (red, n = 9) and Sham (blue, n = 9) as well as their respective contralateral control eyes (C-OHT, unfilled n = 7; C-Sham, gray, = 8). (B) Average Trpv4 expression relative to Hprt (OHT n = 9, C-OHT n = 8, Sham n = 9, C-Sham n = 8). (C) Average Panx1 expression relative to Hprt (OHT n = 9, C-OHT n = 8, Sham n = 9, C-Sham n = 8). (D) Average Adora3 expression relative to Hprt. (E) Average Entpd1 expression relative to Hprt. (F): ratio of P2x7/Adora3. * p = 0.02.
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