Jeremiah K. H. Lim

AAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo

PURPOSE Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.

The Eye As a Biomarker for Alzheimer's Disease

Alzheimers disease (AD) is a progressive neurodegenerative disorder resulting in dementia and eventual death. It is the leading cause of dementia and the number of cases are projected to rise in the next few decades. Pathological hallmarks of AD include the presence of hyperphosphorylated tau and amyloid protein deposition. Currently, these pathological biomarkers are detected either through cerebrospinal fluid analysis, brain imaging or post-mortem. Though effective, these methods are not widely available due to issues such as the difficulty in acquiring samples, lack of infrastructure or high cost. Given that the eye possesses clear optics and shares many neural and vascular similarities to the brain, it offers a direct window to cerebral pathology. These unique characteristics lend itself to being a relatively inexpensive biomarker for AD which carries the potential for wide implementation. The development of ocular biomarkers can have far implications in the discovery of treatments which can improve the quality of lives of patients. In this review, we consider the current evidence for ocular biomarkers in AD and explore potential future avenues of research in this area.

The effect of ageing on ocular blood flow, oxygen tension and retinal function during and after intraocular pressure elevation.

Purpose To investigate the effect of ageing on the recovery of ocular blood flow, intravitreal oxygen tension and retinal function during and after intraocular pressure (IOP) elevation. Methods Long Evans rats (3- and 14-month-old) underwent acute stepwise IOP elevation from 10 to 120 mmHg (5 mmHg steps each 3 minutes). IOP was then returned to baseline and recovery was monitored for 2 hours. Photopic electroretinograms (ERG) were recorded at each IOP step during stress and at each minute during recovery. Ocular blood flow and vitreal oxygen tension (pO2) were assayed continuously and simultaneously using a combined laser Doppler flow meter (LDF) and an oxygen sensitive fibre-optic probe, respectively. The combined sensor was placed in the vitreous chamber, proximal to the retina. Data were binned into 3 minute intervals during stress and 1 min intervals during recovery. Recovery data was described using a bi-logistic function. Results Rats of both ages showed similar susceptibility to IOP elevation, with pO2 showing a closer relationship to ERG than LDF. During recovery, both ages showed a distinctive two-phased recovery for all three measures with the exception of the LDF in 3-month-old rats, which showed only 1 phase. In all animals, LDF recovered fastest (<1 minute), followed by pO2 (<10 minute) and ERG (>1 hour). 14-month-old rats showed surprisingly faster and greater LDF recovery compared to the younger group, with similar levels of pO2 recovery. However, the ERG in these middle-aged animals did not fully recover after two hours, despite showing no difference in susceptibility to IOP during stress compared to the young group. Conclusions Young and middle-aged eyes showed similar susceptibility to IOP elevation in terms of pO2, LDF and ERG. Despite this lack of difference during stress, older eyes did not completely recover function, suggesting a more subtle age-related susceptibility to IOP.

Coupling blood flow and neural function in the retina: a model for homeostatic responses to ocular perfusion pressure challenge

Retinal function is known to be more resistant than blood flow to acute reduction of ocular perfusion pressure (OPP). To understand the mechanisms underlying the disconnect between blood flow and neural function, a mathematical model is developed in this study, which proposes that increased oxygen extraction ratio compensates for relative ischemia to sustain retinal function. In addition, the model incorporates a term to account for a pressure‐related mechanical stress on neurons when OPP reduction is achieved by intraocular pressure (IOP) elevation. We show that this model, combining ocular blood flow, oxygen extraction ratio, and IOP mechanical stress on neurons, accounts for retinal function over a wide range of OPP manipulations. The robustness of the model is tested against experimental data where ocular blood flow, oxygen tension, and retinal function were simultaneously measured during acute OPP manipulation. The model provides a basis for understanding the retinal hemodynamic responses to short‐term OPP challenge.

Retinal biomarkers provide “insight” into cortical pharmacology and disease

&NA; The retina is an easily accessible out‐pouching of the central nervous system (CNS) and thus lends itself to being a biomarker of the brain. More specifically, the presence of neuronal, vascular and blood‐neural barrier parallels in the eye and brain coupled with fast and inexpensive methods to quantify retinal changes make ocular biomarkers an attractive option. This includes its utility as a biomarker for a number of cerebrovascular diseases as well as a drug pharmacology and safety biomarker for the CNS. It is a rapidly emerging field, with some areas well established, such as stroke risk and multiple sclerosis, whereas others are still in development (Alzheimers, Parkinsons, psychological disease and cortical diabetic dysfunction). The current applications and future potential of retinal biomarkers, including potential ways to improve their sensitivity and specificity are discussed. This review summarises the existing literature and provides a perspective on the strength of current retinal biomarkers and their future potential.

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

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.

Longitudinal Changes to Tight Junction Expression and Endothelial Cell Integrity in a Mouse Model of Sterile Corneal Inflammation

PURPOSE We previously reported that applying toll-like receptor (TLR) ligands to an injured cornea induces corneal edema at 24 hours, which subsides by 1 week. We tested the hypotheses that endothelial expression of the tight-junction protein, zonula occludens-1 (ZO-1), would be altered during experimental sterile corneal inflammation and that endothelial cell density (ECD) would remain unaffected. METHODS Anesthetized C57BL/6J mice received central 1-mm corneal abrasions followed by topical application of saline or cytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN, TLR-9 agonist). At 24 hours, 1 week and 4 weeks post treatment, spectral-domain optical coherence tomography images were captured. Eyes were enucleated and processed for zonula occludens-1 (ZO-1) immunofluorescent staining. Corneal flatmounts were analyzed for endothelial ZO-1 expression, cell density, polymegethism, and polymorphism. Corneal stromal inflammatory cell infiltration was evaluated at 4 weeks by immunostaining for CD45. RESULTS Central corneal thickness (CCT) was increased in CpG-ODN treated eyes at 24 hours, had normalized by 1 week, but was again thickened by 4 weeks. In eyes with CpG-ODN, endothelial cell ZO-1 expression was reduced at 24 hours but returned to normal levels by 1 week. Endothelial cell density was not altered at 24 hours or 1 week. By 4 weeks, only CpG-ODN eyes showed relatively reduced ECD, as well as large numbers of CD45+ cells in the stroma. Changes to ECD correlated with CCT (r = -0.53, P < 0.01). Compared with naïve controls, more saline- and CpG-ODN-treated eyes exhibited polymegethism. CONCLUSIONS This study provides novel insights into the interplay between endothelial cell integrity, corneal edema, and chronic stromal leukocyte activation during sterile corneal inflammation in mice.