Eye / Vision

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    P1-CPP promotes Foxo1 and Creb signaling and reduces apoptosis in Neurotrophic Factor-Deprived Primary Retinal Ganglion Cells
    (2023) Johnson, Gretchen; Pham, Jennifer; Krishnamoorthy, Raghu; Nagaraj, Ram; Stankowska, Dorota
    Purpose: To elucidate the intracellular mechanisms underlying neuroprotective effects of the core peptide of a-B crystallin, peptain-1 (P1) conjugated to a cell-permeable peptide CPP (P1-CPP) in primary retinal ganglion cells (RGCs). Targets of the investigation were limited to Creb1, Bak1/Bad, and Foxo1, based upon RNA sequencing data obtained from RGCs of IOP-elevated rats treated with P1-CPP in comparison with the vehicle. Methods: Primary RGCs isolated from Sprague Dawley rat pups were deprived of neurotrophic factors (NT) namely, BDNF, CNTF, and Forskolin for 48 hours, either in the presence or absence of P1-CPP (4µM). After the treatments, RNA isolation was carried out using Trizol reagent. Subsequently, cDNA synthesis and qPCR analysis of the target genes expression, including Creb1 (n=2), Foxo1 (n=3), and Bak1 (n=3), was performed. Another set of RGCs subjected to the same treatments was fixed with 4% paraformaldehyde for 20 minutes and used for immunocytochemical analyses of p-CREB (n=3), FOXO1 (n=3), and BAD (n=3) protein expression. Immunostaining with an RBPMS antibody was used as an RGC marker. N indicates experimental repeats. Results: Following NT deprivation, there was an increase in mRNA expression of Creb1 (2-fold) in RGCs treated with P1-CPP, compared to the vehicle-treated RGCs. Moreover, the phosphorylated (active) form, p-CREB, was increased (by 102%; p=0.04) in primary RGCs treated with P1-CPP, compared to the vehicle-treated group. Pro-apoptotic Bak1 mRNA expression was not changed in the P1-CPP-treated RGCs compared to the vehicle-treated group. Primary RGCs stained for BAD protein showed a decrease (by 62%; p=0.08) in the P1-CPP treated group compared to the vehicle-treated RGCs. Foxo1 mRNA levels were increased by more than 2-fold in the P1-CPP treated RGCs, compared to the vehicle-treated RGCs. FOXO1 protein was also elevated in primary RGCs treated with P1-CPP compared to the vehicle group (by 59%). Conclusions: P1-CPP is neuroprotective against neurotrophic factor deprivation through multiple mechanisms, including early changes in the expression of mitochondrial homeostasis regulator Foxo1, activation of the pro-survival CREB pathway, and inhibition of pro-apoptotic members of the BCL-2 family of proteins.
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    Ketogenic Diet Increases Mitophagy in a Mouse Model of Glaucoma
    (2023) Morgan, Autumn; Fan, Yan; Inman, Denise
    Purpose: We have previously shown that limiting dietary intake to high fat, low protein, and negligible carbohydrate results in mitochondrial biogenesis, and in the case of glaucoma, a reduction in neurodegeneration of retinal ganglion cells (RGCs). In this experimental follow-up study, we wanted to examine the effect of the ketogenic diet on mitophagy, or mitochondrial recycling, within the glaucomatous retina. Methods: MitoQC mice were placed on a ketogenic diet or standard rodent chow for 5 weeks and ocular hypertension (OHT) was induced via microbead injection. The MitoQC reporter mice have a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane that results in retention of red fluorescence when mitochondria bound for recycling are engulfed by lysosomes. The FIJI (ImageJ) macro MitoQC counter was used to quantify red puncta (mitolysosomes) in sectioned retina as a measure of mitophagy within the RGCs and Müller glia. Results: Mitophagy in RGCs, as measured by red puncta, was significantly decreased by ocular hypertension in the control retina (Control + OHT) in comparison to naïve control retina (Ctrl; p<0.0001). The ketogenic diet (KD) resulted in a significant increase in mitolysosomes in RGCs when compared to Ctrl (p<0.0001), Control + OHT (p<0.0001) and KD + OHT (p=0.0089). The ketogenic mice with OHT showed a significantly higher RGC-associated mitolysosome number than Control + OHT mice (p<0.0001). In contrast, mitolysosomes quantified in the Müller glia of Control + OHT mice were significantly higher than the naïve control mice (p=0.0127). Mice in the KD (p=0.0001) and KD + OHT(p=0.0005) groups had significantly greater mitolysosomes than the control Müller glia, however there was no difference in mitophagy between the Control + OHT, KD, and KD + OHT Müller glia groups. Conclusion: Our data demonstrates that mitophagy is managed differently within RGCs and Müller glia of mouse retinas. The KD promoted mitophagy within the RGCs to a degree that overcame the decline of mitophagy after OHT in the control group. Within the Müller glia, the KD was redundant because OHT alone increased mitophagy to similar levels as the KD. These findings suggest a divergence of mitochondrial homeostasis in RGCs and Müller glia that may reflect the different metabolic needs of these cell types.
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    Outcomes of XEN-45 Gel Stent in Open-Angle Glaucoma using the PoST Technique: 12-month Follow-up
    (2023) Liu, Angela; Dhar, Lauren; Hung, Sarah; Fellman, Ronald; Grover, Davinder
    Purpose: The purpose of this study was to report the 12-month surgical outcomes of the novel PoST technique (Posterior Sweep of Tenon’s technique) in eyes undergoing glaucoma surgery with the XEN-45 Gel Stent. Methods: A retrospective review was performed for patients who underwent ab interno implantation of a XEN-45 Gel Stent using the PoST technique with or without concurrent cataract surgery. The surgery was performed in adults with open-angle glaucoma that was refractory to prior surgical treatment and glaucoma medications. Results: In total, 87 eyes of 70 patients aged 58 to 91 years were treated with the XEN-45 Gel Stent. 54 eyes (62%) had a prior glaucoma procedure to lower intraocular pressure (IOP), including laser peripheral iridotomy (LPI). 33 eyes (38%) did not have a prior glaucoma procedure. Across all 87 eyes, the average IOP decrease at 12 months was 6.50 mmHg (28%), from 18.66 mmHg to 12.16 mmHg, with an average decrease of 2.37 (69%) glaucoma medications, from 3.36 to 0.99 anti-glaucoma medications. Patients with a prior glaucoma procedure had an average IOP decrease of 7.84 mmHg (33%) at 12 months with an average decrease of 2.39 (66%) glaucoma medications. In patients without a prior glaucoma procedure, at 12 months, the average IOP decrease was 4.29 mmHg (20%) on an average of 2.33 (74%) fewer medications. An average of 62.76 micrograms of mitomycin C was used during XEN surgery, ranging from 30 to 80 micrograms. Across all eyes, the mean IOP at 12-month follow-up was significantly reduced compared to preoperative IOP (P<0.0001). The number of glaucoma medications was also significantly decreased from baseline (P<0.0001) at 12 months. The average IOP decrease in patients without concurrent cataract surgery was significantly greater than in patients with combined XEN and cataract surgery (8 vs. 4 mmHg; P<0.01). Among all study eyes, there were 28 total complications at or after 1 week post-surgery. The most common types of complication were IOP spike > 12 mmHg (55%), followed by blockage of XEN tip in anterior capsule (21%) and blockage of XEN implant (7%). Patients with at least one previous glaucoma procedure were 4.51 times more likely (OR = 4.51, p= 0.0336) to experience a complication after XEN Gel Stent implantation, compared with patients with no previous glaucoma procedures. The cumulative proportion of failure was 0.35 for patients with a previous glaucoma procedure and 0.21 for patients without any prior glaucoma procedures, totaling 0.3 for all eyes. Eight patients (9%) required needling after XEN implantation. The cumulative proportion of re-operation was 0.22. Conclusions: In this study, the XEN-45 Gel Stent was safe and effective in treating 70% to 80% of patients with refractory glaucoma. The use of the PoST technique provided significant IOP and anti-glaucoma medication reduction compared to pre-operative levels. Additionally, the post-operative needling rate is significantly lower than those previously published. The PoST technique should be considered when using the XEN-45 gel stent in glaucoma patients who have failed previous surgical treatment or are taking multiple glaucoma medications without adequate IOP control.
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    Increased Fibronectin Serotonylation in Stretched Optic Nerve Head Astrocytes
    (2023) Rangan, Rajiv; Clark, Abbot; Tovar-Vidales, Tara
    Purpose: Elevated intraocular pressure contributes to glaucomatous optic nerve degeneration by inducing biomechanical stress at the optic nerve head (ONH), especially in the lamina cribrosa (LC). ONH astrocytes (ONHA) in the LC respond to biomechanical signals through extracellular matrix (ECM) remodeling activities, promoting tissue fibrosis and damage to retinal ganglion cell axons. The enzyme transglutaminase 2 (TG2) plays a role in ECM remodeling, in part, due to its ability to post-translationally modify and cross-link ECM proteins. A unique post-translational modification mediated by TG2 is "serotonylation” - the transamidation of the monoamine serotonin (5-hydroxytryptamine, 5HT) to glutamine residues on proteins. It is speculated that serotonylation contributes to fibrotic tissue remodeling, but this process has not been studied in ocular tissues or in primary glial cells. In this study, we examined changes in the serotonylation of fibronectin (FN; a major ECM glycoprotein) by ONHA after exposure to cyclic stretch. Methods: Primary human ONHA strains (n=3) were exposed to 0-12% cyclic stretch for 24h using a FlexCell FX-6000 system. Cell lysates and conditioned medium samples were collected from stretched and control cells. Serotonylation was assessed by probing for serotonin in samples of FN immunoprecipitated out of conditioned media. Protein levels for potential extra- and intra-cellular mediators of serotonylation were examined using western blotting of concentrated conditioned medium samples and cell lysates, respectively. Results: Serotonylated fibronectin was detected in ONHA. Exposure to stretch increased the amount of fibronectin that was serotonylated by 2.49-fold (p=0.0080). After stretching, extracellular FN levels were not changed. Extracellular TG2 levels were increased by 3.76-fold (p=0.0004). In cell lysates, post-stretch levels of both FN and TG2 were decreased by 5.56-fold (p=0.0181) and 2.51-fold (p=0.0441), respectively. Additionally, serotonin 2A and 2C (5HT2A, 5HT2C) receptor levels were unchanged, and serotonin transporter (SERT) levels decreased by 2.94-fold (p=0.0297). Conclusions: Increased FN serotonylation by TG2 is observed in ONHA after exposure to 24h of 0-12% cyclic stretch. Serotonylation may promote increased FN-crosslinking and fibrotic ECM remodeling, an important feature of glaucomatous pathology. The secreted TG2 – which was elevated in response to stretch – is likely to be the primary mediator of this increased serotonylation. The observed decrease in SERT may lead to increased extracellular 5HT levels, which increases the substrate availability for TG2-mediated serotonylation. Though unchanged, activity at the 5HT2A/C G-coupled protein receptors could increase the availability of intracellular calcium required for TG2 activity. Future experiments will be focused on furthering our understanding of how these proteins may interact to promote serotoynlation.
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    Relationship of Down Syndrome with Keratoconus and Gonadotropins
    (2023) Shrestha, Pawan; Escandon, Paulina; Petersen, Melissa; Hjortdal, Jesper; Ramirez, Lito; Karamichos, Dimitrios
    Purpose: Down syndrome (DS), also known as trisomy 21, is a common genetic disorder of chromosomal nondisjunction. DS has been strongly associated with Keratoconus (KC); however, the exact pathobiology remains unexplored. KC is one of the most significant corneal disorders which is characterized by thinning, cone-shaped protrusion, and steepening of the cornea leading to a significant reduction of visual acuity and even blindness. The aim of this study was to investigate the relationship of DS with KC in the context of gonadotrophic hormones. Methods: This study adhered to the declaration of Helsinki. Fifty-eight healthy controls (29 male, 29 female), one hundred and forty-nine KC (112 male, 37 female), and eighty DS (44 male, 36 female) patients were recruited for this study. Plasma samples were collected from all participants. We investigated the expression of Gonadotrophin-releasing hormone (GnRH) and Follicle stimulating hormone (FSH) using enzyme-linked immunosorbent assay (ELISA). Results: Significant downregulation of GnRH expression was observed in KCs when compared with healthy (p = 0.0006) and DSs (p = 0.00249). GnRH was significantly downregulated in KC and DS males, compared to their healthy counterparts, while no significance was observed in females across all diseases. Significant upregulation of FSH levels was observed in DSs compared to both healthy and KCs (p < 0.0001). FSH expression was also significantly elevated in both DS males and females when compared to healthy and KCs. Conclusions: Our results revealed downregulation of GnRH, but upregulation of FSH in DS participants as compared to KCs. These findings provide new insights into the potential association between DS and KC, and substantiate the role of gonadotropins. Further studies are warranted to further understand the underlying mechanisms and potential implications for the treatment and management of these conditions.
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    Impact of GLUT1 Transporter Knockout in Optic Nerve Head Astrocytes and Retinal Ganglion Cells
    (2023) Gollamudi, Phani Sree Harsha; Inman, Denise
    Abstract Purpose: Astrocytes and axons are the primary constituents of the optic nerve head, the initial site of neurodegeneration in glaucoma. This study was intended to understand the metabolic relationship between astrocytes and RGC axons. We hypothesized that reducing glucose transporter-1 (GLUT1) expression in astrocytes will increase the RGC-associated pathology after ocular hypertension (OHT). Methods: Mice expressing a GLUT1 gene flanked by loxP sites behind the GFAP promoter ("GFAP-GLUT1”) mice were used (n=40) and were divided into 4 groups: GLUT1-knockout+OHT, Control OHT, GLUT1-knockout+No OHT, and Control+No OHT. Baseline and final intra-ocular pressure (IOP), pattern electroretinogram (PERG), and visual evoked potential (VEP) measurements were taken. OHT was induced via magnetic microbead injection into the anterior chamber. Retinas, optic nerves, and brains were collected for retinal ganglion cell (RGC) quantification, anterograde transport analysis, biochemical assays, and protein analysis. Results: Statistically significant increases were noted in the IOP data between mice subjected to OHT and the No OHT groups. OHT led to statistically significant decreases in RGC number, regardless of GLUT1 status. A statistically significant decrease in PERG amplitude was noted in all groups subjected to OHT. Interestingly, GLUT1 knockout PERG amplitude was significantly lower than Control at the outset, suggesting a negative impact on retinal physiology from loss of the GLUT1 in astrocytes. Conclusion: Initial observations indicate glial metabolic homeostasis can impact retinal physiology, but GLUT1 knockout did not appear to negatively impact RGC survival. Ongoing analysis will determine if other structures or functions have been compromised by loss of GLUT1 in astrocytes, as well as provide greater insight into the mechanism of physiological change.
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    Intravitreal Endothelin-1 (ET-1) Injection Reduces Mitophagy in Retinal Ganglion Cells in MitoQC Mice
    (2023) Brooks, Calvin D.; Kodati, Bindu; Inman, Denise; Stankowska, Dorota; Krishnamoorthy, Raghu
    Purpose: The peptide endothelin-1 (ET-1), and its receptors are upregulated in the aqueous humor and retina in animal models of experimentally induced ocular hypertension, and have been shown to have a causative role in retinal ganglion cell (RGC) neurodegeneration. The purpose of this experiment was to assess the role of mitophagy in RGC neurodegeneration following intravitreal ET-1 administration in MitoQC mice. Methods: MitoQC mice (Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl on a C57BL/6 background) at the age of 3 months were used for the study. The mitochondria in these mice display both red and green fluorescence due to expression of a mCherry-GFP tag fused to the mitochondrial targeting sequence of an outer mitochondrial membrane protein, FIS1. When these mitochondria are trafficked to the lysosome for degradation, the green fluorescence is quenched, leaving only the red fluorescence. The MitoQC mice were intravitreally injected in both eyes with either ET-1 (1 nmole) or vehicle (water), and 72 hours following the injections the mice eyes were enucleated and retinal flat mounts were live-imaged using a Zeiss LSM 880 super resolution confocal microscope. Z-stack imaging was used to image the ganglion cell layer. For each Z layer, a threshold algorithm was used to define a region of interest (ROI) that included only areas with red fluorescence, after which red and green fluorescence were quantified for that ROI. Red/green fluorescence intensity was calculated and averaged per image. A red/green ratio larger than 1 is indicative of active mitophagy. This ratio was compared between ET-1 and vehicle-injected mice using a Mann-Whitney test (n=4 eyes per group). Results: At 72 hours after injection with ET-1, the average red/green fluorescence ratio in the RGCs was 0.86, while the vehicle-injected mice had an average red/green ratio of 1.29. These ratios were significantly different (P=0.0003), and the smaller red/green ratio in the ET-1 group indicates lesser mitophagy than the vehicle group. Conclusion: Mitophagy is known to be an important quality control mechanism for neuronal cell survival, and this study provides evidence that mitophagy is impaired by ET-1. The finding indicates that a decline in mitophagy may be associated with endothelin-mediated neurodegeneration in RGCs.
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    Neuroprotection of Rodent Retinal Ganglion Cells using Hybrid Molecule SA-10
    (2023) Pham, Jennifer H.; Kodati, Bindu; Johnson, Gretchen A.; Acharya, Suchismita; Stankowska, Dorota L.
    Purpose: Oxidative stress is the imbalance between the activity of antioxidants and free radical production, which has been shown to be associated with glaucomatous retinal ganglion cell (RGC) degeneration. In this study, we aimed to promote RGC survival by treatment with SA-10, a second-generation hybrid molecule with nitric oxide donating and sulfone reactive oxygen species (ROS) scavenging moieties in vitro and ex vivo following oxidative stress-induced injury. Methods: Endothelin-3, a vasoactive peptide, was used to induce oxidative stress in vitro in rat primary RGCs (n=3 biological replicates) and ex vivo in C57BL/6J mice retinal explants (n=8-9 explants/group). Primary RGCs were isolated from Sprague Dawley rat pups (post-natal days 4-7) and cultured for seven days with neurotrophic factors to allow for neurite outgrowth. The RGCs and retinal explants were pretreated with vehicle (DPBS) or SA-10 [10 µM] for 30 minutes, following which ET-3 treatment [100 nM or 400 nM] was carried out for 1 hour. CellROX™ Green was then used to stain for ROS produced by the cells, and the integrated density was analyzed. Analysis of Variance (ANOVA) or nonparametric Kruskal-Wallis was performed for all experiments. Results: In primary RGCs, ET-3-mediated ROS production decreased by 25.9% (p<0.01) following SA-10 treatment compared to the vehicle. In mice, retinal explants, 400 nM ET-3 induced a 24.4% increase in ROS production compared to the vehicle [0 nM ET-3]. With the SA-10 treatment, the ROS production was decreased by 14.74% (p<0.001) in the ET-3 and SA-10 treated group compared to the ET-3-only treated group. Conclusion: SA-10 effectively protects rodent RGCs in vitro and ex vivo from ET-3-mediated oxidative stress.
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    Effect of Monocarboxylate Transporter 2 Loss on Retinal Ganglion Cell Survival and Function
    (2023) Murinda, Kudakwashe; Morgan, Autumn; Inman, Denise; Kiehlbauch, Charles
    Purpose: There is currently no cure for the vision loss in glaucoma that is characterized by retinal ganglion cell (RGC) loss and irreversible optic neuropathy. Monocarboxylate transporter 2 (MCT2s) that transport pyruvate, lactate, and ketone bodies, are exclusively found in neurons such as the RGCs. We have previously shown that MCT2 is lost during glaucoma, in advance of RGC loss, and MCT2 overexpression protects RGC number and function. This study was undertaken to test whether MCT2s are necessary for RGC survival and function. Methods: To test this hypothesis, we used tamoxifen injection into Thy1-ERT2-cre: MCT2fl/fl mice to conditionally knock out MCT2 from Thy1-positive RGCs. Control mice carried the MCT2 flox’d allele but were Thy1-ERT2-cre-negative. Control and experimental mice were subjected to ocular hypertension using the magnetic microbead model; separate naïve controls from each genotype were also evaluated. Intraocular pressure (IOP) was measured using the TonoLab rebound tonometer. Pattern electroretinogram (PERG) was used to analyze RGC function. We used unbiased stereology (Stereo Investigator, Micro Brightfield) to count the number of retinal ganglion cells in wholemount retina, and ATP levels in retina were also measured. Results: IOP was higher in the ocular hypertension (OHT) groups. MCT2 knockout alone did not impact IOP, nor did it alter baseline PERG amplitude or latency. After OHT, PERG amplitude was significantly lower in the MCT2-knockout mice (p=0.0013). MCT2 knockout alone did not change RGC density. After OHT, RGC density decreased, though in this preliminary analysis, RGC density among the groups was not significantly different. ATP production in the OHT+ Tamoxifen group was significantly higher (1.81 +/- 0.89 ug/ul) than in the naïve control group (0.68 +/- 0.42 ug/ul). Conclusions: MCT2 knockout from RGCs did not change IOP or PERG, suggesting that MCT2 is not necessary for RGC survival. Ocular hypertension decreased PERG amplitude and RGC density, though the magnitude of the decrease may not have been increased by MCT2 knockout. These preliminary data suggest that RGCs are capable of meeting their immediate metabolic needs through means beyond MCT2.
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    Novel functions of the Glutaredoxin (Grx) System in the Lens
    (2023) Zhang, Jinmin; Dang, Terry; Yu, Yu; Wu, Hongli
    Purpose: The purpose of this study is to evaluate the function and therapeutic potential of the glutaredoxin (Grx) system, both glutaredoxin 1 (Grx1) and glutaredoxin 2 (Grx2), using Grx1/Grx2 double knockout (DKO) mice as a model. Methods: We isolated primary LECs from wild-type (WT) and DKO mice for in vitro studies including cell proliferation assays, cell cycle distribution analysis via flow cytometry, cell apoptosis via western blot and ELISA kit, mitochondrial function evaluation via ATP bioluminescence assay, expression levels of mitochondrial complexes I-V, and seahorse mito stress test, cell cytoskeleton visualization using a fluorescence microscope. Results: We found that DKO cells displayed a much slower proliferation rate compared to WT cells. The population of DKO cells in the G2/M phase was two-fold higher than that of WT cells. On the other hand, the population of DKO cells in the S phase was 50% less than that of WT cells. Additionally, DKO cells are pro-apoptotic under non-stressed condition as indicated by higher levels of Bax and cytochrome C. For the mitochondrial function, lower ATP production, less expression of mitochondrial complex III subunit UQCRC2 and complex IV subunit MTCO1 (CIV-MTCO1), lower coupling efficiency, and higher proton leak were presented in DKO cells as compared to WT cells, indicating multi-dimensional mitochondrial dysfunction in DKO cells. As for the cell cytoskeletal organization, we found that DKO cells had microtubule polarization because of the higher levels of vimentin expression which is an indicator of nuclei degeneration inhibition during the lens cell differentiation. Conclusion: Overall, we found slow cell proliferation, cell cycle arrest, and mitochondrial dysfunction in the LECs from DKO mice. Our data indicate Grx system plays an important role in maintaining the normal function of mLECs, and Grx system activation might serve as a new therapeutic strategy for cataract prevention.
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    Novel use of Light Adjustable Lenses to improve visual acuity in patients with corneal abnormalities: a case report.
    (2023) Worley, Josh; Capps, Zachary
    Background: Keratoconus is characterized by thinning and protrusion of the cornea that produces an irregular astigmatism and decreased visual acuity. Individuals with these corneal abnormalities commonly experience undesirable visual acuity outcomes due to lack of effective traditional treatment options. We postulate Light Adjustable Lens (LAL) may be a safe and effective treatment where neither multifocality nor Laser-Assisted In Situ Keratomileusis (LASIK) are a viable method to achieve decreased spectacle dependence following surgery. This report presents a single patient diagnosed with forme fruste keratoconus undergoing LAL intraocular lens (IOL) replacement surgery. The patient was selected based on their history of corneal abnormalities and ability to benefit from IOL replacement. Potential benefit from IOL replacement was determined based on the patient’s ocular complaints and visual test results in clinic. Lens selection parameters were made based on keratotomy and biometry data obtain from the Lenstar LS900. Visual acuities after LAL implantation and postoperative light treatments were recorded and compared with preoperative visual acuities. The patient received LAL, made of foldable silicone, implanted through phacoemulsification and standard IOL implantation techniques in both eyes (OU), and after completion of 1-month postsurgical healing received postoperative light treatments for lens adjustment. Case Presentation: A 69-year-old man presented in clinic with complaints of decreased central vision. His brightness acuity test (BAT) was 20/40 OU and slit lamp examination (SLE) and oxidative stability index (OSI) revealed nuclear sclerotic (NS) cataracts of 2+ grade OU. Scheimpflug imaging (Figure 1.1) showed corneal abnormalities consistent with FFK OU. Imaging measured a topographic irregularly irregular astigmatism of 0.50 D at 115 degrees with a total corneal power (TCP) of 0.44 D at 127 degrees in the right eye (OD) and 0.33 D at 80 degrees with a TCP of 0.32 D at 88 degrees in the left eye (OS). Keratotomy measurements can be found in Table 1. The patient’s primary goal was to achieve reduced spectacle dependency. Preoperative serial refraction established refractive stability OU. RxSight LAL of +20.5 D and +20.0 D were selected for the OD and OS respectively. 1-month UDVA was 20/20 in the OS and UNVA was Jager 5 (20/50) OD. Following 2 postoperative UV light treatments spaced 4 days apart and 1 lock-in UV light treatment after 3 days, the patient maintained UDVA of 20/20 in the OS and achieved UNVA of Jager 1 (20/25) OD. Conclusions: This case shows the potential of LAL to be a reliable and reproducible treatment method that can result in positive visual acuity outcomes in patients with corneal abnormalities. It also demonstrates that in patients with FFK and mild keratoconus, LAL has potential to utilize spherical aberration to successfully achieve extend depth of the focus (EDOF) vision.
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    The Neuroprotective Effects of Hybrid SA-10 Nanoparticles in a Glaucoma Mouse Model of Retinal Ischemia/Reperfusion Injury
    (2023) Le, Kim-Tuyen T.; Zhang, Wei; Pham, Jennifer H.; Kodati, Bindu; Amankwa, Charles E.; Engelland, Rachel E.; Hatfield, Brendon R.; Acharya, Suchismita; Stankowska, Dorota L.
    Purpose: The progression of glaucoma is largely dependent on the gross functionality of retinal ganglion cells (RGCs), which transmit visual signals to the brain. Current treatments for glaucoma focus on lowering intraocular pressure (IOP), yet other risk factors such as oxidative stress and poor blood perfusion also contribute to RGC damage. Furthermore, no treatments exist which revitalize dysfunctional RGCs. One promising neuroprotective agent is SA-10, a hybrid compound with reactive oxygen species (ROS) scavenging sulfone moiety and perfusion-enhancing nitric oxide (NO) donor moiety. This study aims to investigate the in vivoneuroprotective effects of the nanoparticle (NP) formulation of SA-10 (SA-10-NPs) on RGCs in an acute rodent model of ischemia/reperfusion (I/R). Methods: C57BL/6J mice were separated into 3 groups (n= 5-8 per group): Sham control, 1% Blank NPs-treated, and 1% SA-10-NPs. Aside from sham, all groups received 4 μL of different blinded topical pre-treatments: poly(lactic-co-glycolic-acid) (PLGA) nanoparticles suspended PBS for the Blank-NPs group, and 1% SA-10 loaded in PLGA for the SA-10-NPs group. Besides the sham, all groups had their anterior chambers cannulated with normal saline to achieve an elevated IOP of 120 mmHg for 60 minutes. After I/R all groups received 4 μL of their respective treatments 3 times a week over 14 days. Pattern electroretinogram (PERG) and pattern visual evoked potential (PVEP) tests were independently performed both prior to I/R insult (baseline) and after completing the treatment regimen. Mouse eyes were then enucleated. Their retinas were stained with an RNA binding protein with multiple splicing (RBPMS) RGC-specific marker for quantification of cell survival. Parametric Analysis of Variance (ANOVA) and its non-parametric equivalent, the Kruskal-Wallis test, were performed for statistical analysis. Results: I/R injury (Blank-NPs-treated) produced a 52.1% decline (p<0.01) in PERG and a 17.9% decreasing trend in PVEP amplitudes as compared to sham. SA-10-NPs prevented this decline by a trend of 33.5% and 14%, respectively. I/R injury (Blank NPs-treated) caused a 33% decrease (p<0.01) in RGC survival in the inner retina in comparison with sham control, which was alleviated with the SA-10-NPs treatment by 33.4% (p<0.001). Conclusions: SA-10-NPs enhanced RGC survival and function following ischemia-induced damage in the mice I/R model and have the potential to be used as a neuroprotective therapy for glaucoma.
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    Evaluating the properties of extracellular vesicles that are affected by diabetic keratopathy.
    (2023) Hefley, Brenna; Shrestha, Pawan; Ma, Jian-Xing; Karamichos, Dimitrios
    Purpose: Extracellular Vesicles (EVs) are small-sphere like structures that are released from cells. They play a role in cell-cell communication by passing genetic information from one cell to another. EVs are thought to be critical in future diagnostic targets for various diseases and conditions. Diabetic Keratopathy (DK) can often lead to different manifestations such as scarring and corneal erosions. One of the receptors that plays a role in DK is Peroxisome Proliferator-Activated Receptor alpha (PPARα), but its functions in the cornea is unknown, including its impact on the EVs formation. Our goal was to investigate the influence of PPARα in the production of EVs and its role in cell-cell interaction. Methods: Healthy Corneal Fibroblasts (HCFs), Type 1 Diabetes Mellitus (T1DM), and Type 2 Diabetes Mellitus (T2DM) corneal stromal cells were cultured on polycarbonate membranes for 4 weeks at a density of 1x106 cell/well in culture medium + 0.5mM stable Vitamin C. The culture media were processed and analyzed with the EV-TETRA-C chips paired with the ExoView R100. Results: The results showed that total particle counts were upregulated in HCF compared to T1DM and T2DM, but were downregulated in T1DM when compared with T2DM, during weeks 2 and 4. The total particle count for HCFs were downregulated during week 4 when compared to weeks 1 and 3. When comparing week 1 to weeks 2, 3, and 4, week 1 revealed the most significance for changes of CD63+, CD63+/CD81+, CD81+/CD9+, and CD63+/CD81+/CD9+ in HCFs and T1DMs. CD9+ showed significance during weeks 2, 3, and 4 in HCFs and T1DMs. During week 1, T2DMs had significant downregulation of CD63+, whereas CD9+, CD63+/CD9+, and CD63+/CD81+/CD9+ showed significant upregulation. During week 4, T2DMs showed significant downregulation in CD63+/CD81+ and significant upregulation of CD81+/CD9+. T1DMs were the only cell type to show significance in CD81+, which was during week 4 when compared to week 2. CD63+, CD81+, and CD9+ showed significance during week 1 and 2 in HCF, T1DM, and T2DM. The co/triple colocalizations showed significance in weeks 1, 2, and 4 in HCF, T1DM, and T2DM. Conclusion: EV formation (based on CD63+), immune system regulation (CD81+ and CD9+), and EV particle counts showed distinct phenotypes between HCF, T1DM, and T2DM. These distinctions could potentially serve as a diagnostic tool in the future and ultimately help individuals suffering from DK.
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    Development of Reconstituted High-Density Lipoprotein Nanoparticles Utilizing Fluorescence Resonance Energy Transfer for Ocular Applications
    (2023) Petty, R. Max
    Purpose: Macular degeneration and glaucoma are considered age-related degenerative eye diseases. Both conditions can lead to vision change and loss. While glaucoma and macular degeneration have similarities, they affect different eye regions and require targeted drug-delivery systems. Significant limitations of current ocular therapies are poor bioavailability and delivery barriers in the eye. Developing an efficient ocular delivery system is thus critical to improving the efficacy of therapeutic agents. Specifically, reconstituted high-density lipoprotein (rHDL) mimics the structure and function of endogenous human plasma HDL and thus presents a non-toxic therapeutic strategy for delivering various drugs and imaging agents to ocular tissue. Moreover, rHDL nanoparticles (rHDL NPs) are ideal for transporting lipophilic therapeutic agents and imaging dyes since they are small in size, non-immunogenic, can circulate in the body fluids for an extended time, and have specific receptor-protein interactions to release their lipophilic payloads. Our study aims to employ a reconstituted rHDL drug delivery vehicle that mimics the structure and function of endogenous human plasma HDL and offers a novel strategy for the delivery of drugs and imaging agents to the eye. Methods: A stable rHDL-payload complex (rHDL NPs) was prepared by combining lipophilic fluorescent dyes using phosphatidylcholine and apolipoprotein A-I (Apo A-I) via a novel preparation method. Dual fluorescent rHDL NPs have been used as Förster resonance energy transfer (FRET) probes and were assessed by dynamic light scattering (DLS), spectrophotometry, and fluorescence spectroscopy. Results: Dual fluorescence rHDL NPs were generated with 64.4% and 79.2% encapsulation efficiency for the donor and acceptor fluorophores, respectively. rHDL NPs were found to have a polydispersity index (PDI) of 0.302 ± 0.023, an average size of 10.96 ± 1.47 nm, and a zeta potential of -7.65 ± 0.63 mV. The fluorescent signals were characterized by anisotropy measurements while the FRET signal was detected by the change in fluorescence lifetime between the donor and acceptor fluorophores. Conclusions: A stable rHDL NP formulation that includes a FRET pair was successfully prepared through an optimized protocol. The rHDL NPs can be utilized for biodistribution studies and dynamic kinetic characterization in vivo to assess the efficacy of drug-loaded rHDL NPs for the treatment of ocular degenerative diseases such as glaucoma and macular degeneration.