Anatomy and Neuroscience - Research Publications
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ItemNo Preview AvailableReversibility of retinal ganglion cell dysfunction due to chronic IOP elevation.(Association for Research in Vision and Ophthalmology, 2018-07-01)Purpose : To determine the duration of chronic IOP elevation beyond which ganglion cell function can no longer recover using the mouse circumlimbal suture model. Methods : IOP elevation was induced in anaesthetized (isoflurane) adult male C57BL6/J mice by attaching a circumlimbal suture (nylon, 10/0) around the equator of one eye, with the contralateral eye serving as a control. The suture was left in place for 8, 12 and 16 weeks (n=27, 23 and 27), respectively, and animals underwent electroretinography and optical coherence tomography at these time points. In two other groups, the suture was removed after 8 and 12 weeks (n=26 and 28), and the capacity for recovery assessed 4 weeks later. IOP was measured weekly (Tonolab). Retinal ganglion cell (RGC) function (or integrity) was assessed with the positive scotopic threshold response (pSTR) and retinal nerve fibre layer (RNFL) thickness. Data (mean ± SEM) were compared using t-test (control vs. treatment) and one-way ANOVA (within groups). Results : IOP in sutured eyes was higher than control eyes (8wk: 17.1 ± 0.3 vs. 26.8 ± 0.6 mmHg, 12wk: 13.8 ± 0.3 vs. 19.5 ± 0.5 mmHg, 16wk: 17.1 ± 0.2 vs. 27.4 ± 0.6 mmHg; all P<0.001). After suture removal, IOP returned to levels comparable to control eyes (8+4wk: 16.9 ± 0.3 vs. 16.1 ± 0.3 mmHg; P=0.08, 12+4wk: 17.3 ± 0.2 vs. 17.1 ± 0.3 mmHg; P=0.5). With IOP elevation, RGC function declined to 75% ± 8% (8wk), 78% ± 7% (12wk) and 59% ± 4% (16wk, all P<0.001) of control eyes. RNFL thinning was also evident (8wk: 84% ± 4%, 12wk: 83% ± 5%; 16wk: 83% ± 3%; P<0.001) but no change in total retinal thickness was noted (P=0.33). Suture removal at week 8 facilitated full recovery of RGC function (97% ± 7%, P=0.9 vs. baseline) 4 weeks later. However, there was no recovery in RNFL thickness (87% ± 3%, P<0.001 vs. baseline). When the suture was removed at week 12, neither function (79% ± 9%, P<0.05) nor RNFL thickness recovered (89% ± 3%, P<0.01) 4 weeks later. Conclusions : RGC dysfunction can be recovered 4 weeks after an 8-week period of mild IOP elevation, but not after a 12-week period. Beyond 12 weeks, IOP reversal only served to prevent further functional decline. This identifies a critical chronic IOP duration that results in irreversible ganglion cell dysfunction. This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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ItemNo Preview AvailableThe effect of TrkB receptor knockdown on mouse retinal ganglion cell function and their response to acute mild intraocular pressure stress(Association for Research in Vision and Ophthalmology, 2019-07-01)Purpose : To examine the effect of tropomyosin receptor kinase B (TrkB) receptor removal on basal retinal ganglion cell (RGC) function and recovery from acute intraocular pressure (IOP) stress following conditional deletion of neuronal-specific TrkB receptors in adult mice. Methods : Conditional TrkB receptor knockout (KO) from Thy-1 positive neurons was induced via daily tamoxifen injections (100 ul i.p. 75mg/kg, 5 days) in 3-month old Thy1-GFP CreERT2+/- TrkBfl/fl transgenic (TrkB KO n=12) and control CreERT2-/- TrkBfl/fl mice (n=12). Four weeks later, one eye was exposed to controlled IOP stress of 50mmHg for 30 minutes, achieved via anterior chamber cannulation (50 μm glass micropipette connected to height-adjustable Hanks balanced salt solution reservoir; 80:10mg/kg ketamine:xylazine). After 7 days of recovery, retinal function (full-field electroretinogram -5.53 – 2.07 log cd.s/m2) and structure (optical coherence tomography) were assessed in sedated mice. Following in vivo assays, eyes were enucleated for immunohistochemical assessment of TrkB receptor KO efficiency using confocal microscopy. Unpaired t-test and two-way ANOVA were used for statistical analysis. Results : TrkB receptor expression was largely confined to the ganglion cell layers and reduced by 81.3±5.8% in TrkB KO retinas compared to controls (P<0.05). Deletion of TrkB receptors significantly reduced RGC-mediated negative scotopic threshold response (nSTR -39.1±13.7% P<0.05, positive STR -38.0±12.1% P=0.05). No changes in photoreceptor (amplitude P>0.05, sensitivity P>0.05) and bipolar cell (amplitude P>0.05, sensitivity P>0.05) function. At day 7 post-IOP stress, photoreceptor and bipolar cell responses recovered back to baseline whilst RGC function did not (pSTR P<0.05; nSTR P<0.05). This effect was similar for both genotypes. TrkB KO did not affect total retinal, retinal nerve fibre, ganglion cell and inner plexiform layer thicknesses compared with control retina (P>0.05). Conclusions : Conditional removal of TrkB receptors in adult mice suggests that TrkB is critical for the ongoing maintenance of ganglion cell function. Specific changes in RGC morphology, synapse expression or intrinsic excitability associated with TrkB deficiency remain to be elucidated. It appears that TrkB receptors do not play an integral role in recovery from a single episode of mild IOP stress.
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ItemReversibility of Retinal Ganglion Cell Dysfunction From Chronic IOP Elevation(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2019-09)PURPOSE: To test the hypothesis that the capacity for retinal ganglion cells to functionally recover from chronic IOP elevation is dependent on the duration of IOP elevation. METHODS: IOP elevation was induced in one eye in anesthetized (isoflurane) adult C57BL6/J mice using a circumlimbal suture. Sutures were left in place for 8 and 16 weeks (n = 30 and 28). In two other groups the suture was cut after 8 and 12 weeks (n = 30 and 28), and ganglion cell function (electroretinography) and retinal structure (optical coherence tomography) were assessed 4 weeks later. Ganglion cell density was quantified by counting RBPMS (RNA-binding protein with multiple splicing)-stained cells. RESULTS: With IOP elevation (∼10 mm Hg above baseline), ganglion cell function declined to 75% ± 8% at 8 weeks and 59% ± 4% at 16 weeks relative to contralateral control eyes. The retinal nerve fiber layer was thinner at 8 (84% ± 4%) and 16 weeks (83% ± 3%), without a significant difference in total retinal thickness. Ganglion cell function recovered with IOP normalization (suture removal) at week 8 (97% ± 7%), but not at week 12 (73% ± 6%). Ganglion cell loss was found in all groups (-8% to -13%). CONCLUSIONS: In the mouse circumlimbal suture model, 12 weeks of IOP elevation resulted in irreversible ganglion cell dysfunction, whereas retinal dysfunction was fully reversible after 8 weeks of IOP elevation.
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ItemCharacterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels.(Frontiers Media SA, 2017)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.